Using truncated guide RNAs (tru-gRNAs) to increase specificity for RNA-guided genome editing

ABSTRACT

Methods for increasing specificity of RNA-guided genome editing, e.g., editing using CRISPR/Cas9 systems, using truncated guide RNAs (tru-gRNAs).

CLAIM OF PRIORITY

This application claims the benefit of U.S. Patent Application Ser. Nos. 61/799,647, filed on Mar. 15, 2013; 61/838,178, filed on Jun. 21, 2013; 61/838,148, filed on Jun. 21, 2013, and 61/921,007, filed on Dec. 26, 2013. The entire contents of the foregoing are hereby incorporated by reference.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Grant Nos. GM105189, GM105378, GM088040, AR063070, HG005550, awarded by the National Institutes of Health and Grant No. W911NF-11-2-0056 awarded by the U.S. Department of Army. The Government has certain rights in the invention.

TECHNICAL FIELD

Methods for increasing specificity of RNA-guided genome editing, e.g., editing using CRISPR/Cas9 systems, using truncated guide RNAs (tru-gRNAs).

BACKGROUND

Recent work has demonstrated that clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems (Wiedenheft et al., Nature 482, 331-338 (2012); Horvath et al., Science 327, 167-170 (2010); Terns et al., Curr Opin Microbiol 14, 321-327 (2011)) can serve as the basis for performing genome editing in bacteria, yeast and human cells, as well as in vivo in whole organisms such as fruit flies, zebrafish and mice (Wang et al., Cell 153, 910-918 (2013); Shen et al., Cell Res (2013); Dicarlo et al., Nucleic Acids Res (2013); Jiang et al., Nat Biotechnol 31, 233-239 (2013); Jinek et al., Elife 2, e00471 (2013); Hwang et al., Nat Biotechnol 31, 227-229 (2013); Cong et al., Science 339, 819-823 (2013); Mali et al., Science 339, 823-826 (2013c); Cho et al., Nat Biotechnol 31, 230-232 (2013); Gratz et al., Genetics 194(4):1029-35 (2013)). The Cas9 nuclease from S. pyogenes (hereafter simply Cas9) can be guided via base pair complementarity between the first 20 nucleotides of an engineered guide RNA (gRNA) and the complementary strand of a target genomic DNA sequence of interest that lies next to a protospacer adjacent motif (PAM), e.g., a PAM matching the sequence NGG or NAG (Shen et al., Cell Res (2013); Dicarlo et al., Nucleic Acids Res (2013); Jiang et al., Nat Biotechnol 31, 233-239 (2013); Jinek et al., Elife 2, e00471 (2013); Hwang et al., Nat Biotechnol 31, 227-229 (2013); Cong et al., Science 339, 819-823 (2013); Mali et al., Science 339, 823-826 (2013c); Cho et al., Nat Biotechnol 31, 230-232 (2013); Jinek et al., Science 337, 816-821 (2012)). Previous studies performed in vitro (Jinek et al., Science 337, 816-821 (2012)), in bacteria (Jiang et al., Nat Biotechnol 31, 233-239 (2013)) and in human cells (Cong et al., Science 339, 819-823 (2013)) have shown that Cas9-mediated cleavage can, in some cases, be abolished by single mismatches at the gRNA/target site interface, particularly in the last 10-12 nucleotides (nts) located in the 3′ end of the 20 nt gRNA complementarity region.

SUMMARY

CRISPR-Cas genome editing uses a guide RNA, which includes both a complementarity region (which binds the target DNA by base-pairing) and a Cas9-binding region, to direct a Cas9 nuclease to a target DNA (see FIG. 1). The nuclease can tolerate a number of mismatches (up to five, as shown herein) in the complementarity region and still cleave; it is hard to predict the effects of any given single or combination of mismatches on activity. Taken together, these nucleases can show significant off-target effects but it can be challenging to predict these sites. Described herein are methods for increasing the specificity of genome editing using the CRISPR/Cas system, e.g., using Cas9 or Cas9-based fusion proteins. In particular, provided are truncated guide RNAs (tru-gRNAs) that include a shortened target complementarity region (i.e., less than 20 nts, e.g., 17-19 or 17-18 nts of target complementarity, e.g., 17, 18 or 19 nts of target complementarity), and methods of using the same. As used herein, “17-18 or 17-19” includes 17, 18, or 19 nucleotides.

In one aspect, the invention provides a guide RNA molecule (e.g., a single guide RNA or a crRNA) having a target complementarity region of 17-18 or 17-19 nucleotides, e.g., the target complementarity region consists of 17-18 or 17-19 nucleotides, e.g., the target complementarity region consists of 17-18 or 17-19 nucleotides of consecutive target complementarity. In some embodiments, the guide RNA includes a complementarity region consisting of 17-18 or 17-19 nucleotides that are complementary to 17-18 or 17-19 consecutive nucleotides of the complementary strand of a selected target genomic sequence. In some embodiments, the target complementarity region consists of 17-18 nucleotides (of target complementarity). In some embodiments, the complementarity region is complementary to 17 consecutive nucleotides of the complementary strand of a selected target sequence. In some embodiments, the complementarity region is complementary to 18 consecutive nucleotides of the complementary strand of a selected target sequence.

In another aspect, the invention provides a ribonucleic acid consisting of the sequence:

(SEQ ID NO: 2404) (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUA; (SEQ ID NO: 2407) (X₁₇₋₁₈ or X₁₇₋₁₉) GUUUUAGAGCUAUGCUGUUUUG; or (SEQ ID NO: 2408) (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUAUGCU; (SEQ ID NO: 1) (X₁₇₋₁₈ or X₁₇₋₁₉) GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCG(X_(N)); (SEQ ID NO: 2) (X₁₇₋₁₈ or X₁₇₋₁₉) GUUUUAGAGCUAUGCUGAAAAGCAUAGCAAGUUAAAAUAAGGCU AGUCCGUUAUC(X_(N)); (SEQ ID NO: 3) (X₁₇₋₁₈ or X₁₇₋₁₉) GUUUUAGAGCUAUGCUGUUUUGGAAACAAAACAGCAUAGCAAGU UAAAAUAAGGCUAGUCCGUUAUC(X_(N)); (SEQ ID NO: 4) (X₁₇₋₁₈) GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUU AUCAACUUGAAAAAGUGGCACCGAGUCGGUGC(X_(N)), (SEQ ID NO: 5) (X₁₇₋₁₈ or X₁₇₋₁₉) GUUUAAGAGCUAGAAAUAGCAAGUUUAAAUAAGGCUAGUCCGUU AUCAACUUGAAAAAGUGGCACCGAGUCGGUGC; (SEQ ID NO: 6) (X₁₇₋₁₈ or X₁₇₋₁₉) GUUUUAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGG CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC; or (SEQ ID NO: 7) (X₁₇₋₁₈ or X₁₇₋₁₉) GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGG CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC; wherein X₁₇₋₁₈ or X₁₇₋₁₉ is a sequence (of 17-18 or 17-19 nucleotides) complementary to the complementary strand of a selected target sequence, preferably a target sequence immediately 5′ of a protospacer adjacent motif (PAM), e.g., NGG, NAG, or NNGG (see, for example, the configuration in FIG. 1), and X_(N) is any sequence, wherein N (in the RNA) can be 0-200, e.g., 0-100, 0-50, or 0-20, that does not interfere with the binding of the ribonucleic acid to Cas9. In no case is the X₁₇₋₁₈ or X₁₇₋₁₉ identical to a sequence that naturally occurs adjacent to the rest of the RNA. In some embodiments the RNA includes one or more U, e.g., 1 to 8 or more Us (e.g., U, UU, UUU, UUUU, UUUUU, UUUUUU, UUUUUUU, UUUUUUUU) at the 3′ end of the molecule, as a result of the optional presence of one or more Ts used as a termination signal to terminate RNA PolIII transcription. In some embodiments the RNA includes one or more, e.g., up to 3, e.g., one, two, or three, additional nucleotides at the 5′ end of the RNA molecule that is not complementary to the target sequence. In some embodiments, the target complementarity region consists of 17-18 nucleotides (of target complementarity). In some embodiments, the complementarity region is complementary to 17 consecutive nucleotides of the complementary strand of a selected target sequence. In some embodiments, the complementarity region is complementary to 18 consecutive

In another aspect, the invention provides DNA molecules encoding the ribonucleic acids described herein, and host cells harboring or expressing the ribonucleic acids or vectors.

In a further aspect, the invention provides methods for increasing specificity of RNA-guided genome editing in a cell, the method comprising contacting the cell with a guide RNA that includes a complementarity region consisting of 17-18 or 17-19 nucleotides that are complementary to 17-18 or 17-19 consecutive nucleotides of the complementary strand of a selected target genomic sequence, as described herein.

In yet another aspect, the invention provides methods for inducing a single or double-stranded break in a target region of a double-stranded DNA molecule, e.g., in a genomic sequence in a cell. The methods include expressing in or introducing into the cell: a Cas9 nuclease or nickase; and a guide RNA that includes a sequence consisting of 17 or 18 or 19 nucleotides that are complementary to the complementary strand of a selected target sequence, preferably a target sequence immediately 5′ of a protospacer adjacent motif (PAM), e.g., NGG, NAG, or NNGG, e.g., a ribonucleic acid as described herein.

Also provided herein are methods for modifying a target region of a double-stranded DNA molecule in a cell. The methods include expressing in or introducing into the cell: a dCas9-heterologous functional domain fusion protein (dCas9-HFD); and a guide RNA that includes a complementarity region consisting of 17-18 or 17-19 nucleotides that are complementary to 17-18 or 17-19 consecutive nucleotides of the complementary strand of a selected target genomic sequence, as described herein.

In some embodiments, the guide RNA is (i) a single guide RNA that includes a complementarity region consisting of 17-18 or 17-19 nucleotides that are complementary to 17-18 or 17-19 consecutive nucleotides of the complementary strand of a selected target genomic sequence, or (ii) a crRNA that includes a complementarity region consisting of 17-18 or 17-19 nucleotides that are complementary to 17-18 or 17-19 consecutive nucleotides of the complementary strand of a selected target genomic sequence, and a tracrRNA.

In some embodiments, the target complementarity region consists of 17-18 nucleotides (of target complementarity). In some embodiments, the complementarity region is complementary to 17 consecutive nucleotides of the complementary strand of a selected target sequence. In some embodiments, the complementarity region is complementary to 18 consecutive

In no case is the X₁₇₋₁₈ or X₁₇₋₁₉ of any of the molecules described herein identical to a sequence that naturally occurs adjacent to the rest of the RNA. In some embodiments the RNA includes one or more U, e.g., 1 to 8 or more Us (e.g., U, UU, UUU, UUUU, UUUUU, UUUUUU, UUUUUUU, UUUUUUUU) at the 3′ end of the molecule, as a result of the optional presence of one or more Ts used as a termination signal to terminate RNA PolIII transcription. In some embodiments the RNA includes one or more, e.g., up to 3, e.g., one, two, or three, additional nucleotides at the 5′ end of the RNA molecule that is not complementary to the target sequence.

In some embodiments, one or more of the nucleotides of the RNA is modified, e.g., locked (2′-O-4′-C methylene bridge), is 5′-methylcytidine, is 2′-O-methyl-pseudouridine, or in which the ribose phosphate backbone has been replaced by a polyamide chain, e.g., one or more of the nucleotides within or outside the target complementarity region X₁₇₋₁₈ or X₁₇₋₁₉. In some embodiments, some or all of the tracrRNA or crRNA, e.g., within or outside the X₁₇₋₁₈ or X₁₇₋₁₉ target complementarity region, comprises deoxyribonucleotides (e.g., is all or partially DNA, e.g. DNA/RNA hybrids).

In an additional aspect, the invention provides methods for modifying a target region of a double-stranded DNA molecule, e.g., in a genomic sequence in a cell. The methods include expressing in or introducing into the cell: a dCas9-heterologous functional domain fusion protein (dCas9-HFD); and a guide RNA that includes a sequence consisting of 17-18 or 17-19 nucleotides that are complementary to the complementary strand of a selected target sequence, preferably a target sequence immediately 5′ of a protospacer adjacent motif (PAM), e.g., NGG, NAG, or NNGG, e.g., a ribonucleic acid as described herein. In no case is the X₁₇₋₁₈ or X₁₇₋₁₉ identical to a sequence that naturally occurs adjacent to the rest of the RNA. In some embodiments the RNA includes one or more, e.g., up to 3, e.g., one, two, or three, additional nucleotides at the 5′ end of the RNA molecule that is not complementary to the target sequence.

In another aspect, the invention provides methods for modifying, e.g., introducing a sequence specific break into, a target region of a double-stranded DNA molecule, e.g., in a genomic sequence in a cell. The methods include expressing in or introducing into the cell: a Cas9 nuclease or nickase, or a dCas9-heterologous functional domain fusion protein (dCas9-HFD);

a tracrRNA, e.g., comprising or consisting of the sequence GGAACCAUUCAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUA UCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO:8) or an active portion thereof;

UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGC (SEQ ID NO:2405) or an active portion thereof;

AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGU GGCACCGAGUCGGUGC (SEQ ID NO:2407) or an active portion thereof;

CAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA AAAAGUGGCACCGAGUCGGUGC (SEQ ID NO:2409) or an active portion thereof;

UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG (SEQ ID NO:2410) or an active portion thereof;

UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCA (SEQ ID NO:2411) or an active portion thereof; or

UAGCAAGUUAAAAUAAGGCUAGUCCG (SEQ ID NO:2412) or an active portion thereof; and

a crRNA that includes a sequence consisting of 17-18 or 17-19 nucleotides that are complementary to the complementary strand of a selected target sequence, preferably a target sequence immediately 5′ of a protospacer adjacent motif (PAM), e.g., NGG, NAG, or NNGG; in some embodiments the crRNA has the sequence:

(SEQ ID NO: 2404) (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUA; (SEQ ID NO: 2407) (X₁₇₋₁₈ or X₁₇₋₁₉) GUUUUAGAGCUAUGCUGUUUUG; or (SEQ ID NO: 2408) (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUAUGCU.

In some embodiments the crRNA is (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUAUGCUGUUUUG (SEQ ID NO:2407) and the tracrRNA is GGAACCAUUCAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUA UCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO:8); the cRNA is (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUA (SEQ ID NO:2404) and the tracrRNA is UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGC (SEQ ID NO:2405); or the cRNA is (X₁₇₋₁₈ or X₁₇₋₁₉) GUUUUAGAGCUAUGCU (SEQ ID NO:2408) and the tracrRNA is AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGU GGCACCGAGUCGGUGC (SEQ ID NO:2406).

In no case is the X₁₇₋₁₈ or X₁₇₋₁₉ identical to a sequence that naturally occurs adjacent to the rest of the RNA. In some embodiments the RNA (e.g., tracrRNA or crRNA) includes one or more U, e.g., 2 to 8 or more Us (e.g., U, UU, UUU, UUUU, UUUUU, UUUUUU, UUUUUUU, UUUUUUUU) at the 3′ end of the molecule, as a result of the optional presence of one or more Ts used as a termination signal to terminate RNA PolIII transcription. In some embodiments the RNA (e.g., tracrRNA or crRNA) includes one or more, e.g., up to 3, e.g., one, two, or three, additional nucleotides at the 5′ end of the RNA molecule that is not complementary to the target sequence. In some embodiments, one or more of the nucleotides of the crRNA or tracrRNA is modified, e.g., locked (2′-O-4′-C methylene bridge), is 5′-methylcytidine, is 2′-O-methyl-pseudouridine, or in which the ribose phosphate backbone has been replaced by a polyamide chain, e.g., one or more of the nucleotides within or outside the sequence X₁₇₋₁₈ or X₁₇₋₁₉. In some embodiments, some or all of the tracrRNA or crRNA, e.g., within or outside the X₁₇₋₁₈ or X₁₇₋₁₉ target complementarity region, comprises deoxyribonucleotides (e.g., is all or partially DNA, e.g. DNA/RNA hybrids).

In some embodiments, the dCas9-heterologous functional domain fusion protein (dCas9-HFD) comprises a HFD that modifies gene expression, histones, or DNA, e.g., transcriptional activation domain, transcriptional repressors (e.g., silencers such as Heterochromatin Protein 1 (HP1), e.g., HP1α or HP1β), enzymes that modify the methylation state of DNA (e.g., DNA methyltransferase (DNMT) or TET proteins, e.g., TET1), or enzymes that modify histone subunit (e.g., histone acetyltransferases (HAT), histone deacetylases (HDAC), or histone demethylases). In preferred embodiments, the heterologous functional domain is a transcriptional activation domain, e.g., a VP64 or NF-κB p65 transcriptional activation domain; an enzyme that catalyzes DNA demethylation, e.g., a TET protein family member or the catalytic domain from one of these family members; or histone modification (e.g., LSD1, histone methyltransferase, HDACs, or HATs) or a transcription silencing domain, e.g., from Heterochromatin Protein 1 (HP1), e.g., HP1α or HP1β; or a biological tether, e.g., MS2, CRISPR/Cas Subtype Ypest protein 4 (Csy4) or lambda N protein. dCas9-HFD are described in a U.S. Provisional Patent Applications U.S. Ser. No. 61/799,647, Filed on Mar. 15, 2013, U.S. Ser. No. 61/838,148, filed on Jun. 21, 2013, and PCT International Application No. PCT/US14/27335, all of which are incorporated herein by reference in its entirety.

In some embodiments, the methods described herein result in an indel mutation or sequence alteration in the selected target genomic sequence.

In some embodiments, the cell is a eukaryotic cell, e.g., a mammalian cell, e.g., a human cell.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1: Schematic illustrating a gRNA/Cas9 nuclease complex bound to its target DNA site. Scissors indicate approximate cleavage points of the Cas9 nuclease on the genomic DNA target site (SEQ ID NOs: 2691-2692). Note the numbering of nucleotides on the guide RNA (SEQ ID NO: 2693) proceeds in an inverse fashion from 5′ to 3′.

FIG. 2A: Schematic illustrating a rationale for truncating the 5′ complementarity region of a gRNA. Thick grey lines=target DNA site, thin dark grey line structure=gRNA, black lines show base pairing (or lack thereof) between gRNA and target DNA site.

FIG. 2B: Schematic overview of the EGFP disruption assay. Repair of targeted Cas9-mediated double-stranded breaks in a single integrated EGFP-PEST reporter gene by error-prone NHEJ-mediated repair leads to frame-shift mutations that disrupt the coding sequence and associated loss of fluorescence in cells.

FIGS. 2C-F: Activities of RNA-guided nucleases (RGNs) harboring single guide RNAs (gRNAs) bearing (C) single mismatches, (D) adjacent double mismatches, (E) variably spaced double mismatches, and (F) increasing numbers of adjacent mismatches assayed on three different target sites in the EGFP reporter gene sequence. Mean activities of replicates are shown, normalized to the activity of a perfectly matched single gRNA. Error bars indicate standard errors of the mean. Positions mismatched in each single gRNA are highlighted in grey in the grid below. Sequences of the three EGFP target sites were as follows:

(SEQ ID NO: 9) EGFP Site 1 GGGCACGGGCAGCTTGCCGGTGG (SEQ ID NO: 10) EGFP Site 2 GATGCCGTTCTTCTGCTTGTCGG (SEQ ID NO: 11) EGFP Site 3 GGTGGTGCAGATGAACTTCAGGG

FIG. 2G: Mismatches at the 5′ end of the gRNA make CRISPR/Cas more sensitive more 3′ mismatches. The gRNAs Watson-Crick base pair between the RNA&DNA with the exception of positions indicated with an “m” which are mismatched using the Watson-Crick transversion (i.e., EGFP Site#2 M18-19 is mismatched by changing the gRNA to its Watson-Crick partner at positions 18 & 19. Although positions near the 5′ of the gRNA are generally very well tolerated, matches in these positions are important for nuclease activity when other residues are mismatched. When all four positions are mismatched, nuclease activity is no longer detectable. This further demonstrates that matches at these 5′ position can help compensate for mismatches at other more 3′ positions. Note these experiments were performed with a non-codon optimized version of Cas9 which can show lower absolute levels of nuclease activity as compared to the codon optimized version.

FIG. 2H: Efficiency of Cas9 nuclease activities directed by gRNAs bearing variable length complementarity regions ranging from 15 to 25 nts in a human cell-based U2OS EGFP disruption assay. Expression of a gRNA from the U6 promoter requires the presence of a 5′ G and therefore it was only possible to evaluate gRNAs harboring certain lengths of complementarity to the target DNA site (15, 17, 19, 20, 21, 23, and 25 nts) (SEQ ID NOs: 2694-2700, respectively, in order of appearance).

FIG. 3A: Efficiencies of EGFP disruption in human cells mediated by Cas9 and full-length or shortened gRNAs for four target sites in the EGFP reporter gene. Lengths of complementarity regions and corresponding target DNA sites (SEQ ID NOs: 2701, 9, 2702, 10, 2703-2704, 11 and 2705-2707, respectively, in order of appearance) are shown. Ctrl=control gRNA lacking a complementarity region.

FIG. 3B: Efficiencies of targeted indel mutations introduced at seven different human endogenous gene targets by matched standard RGNs (Cas9 and standard full-length gRNAs) and tru-RGNs (Cas9 and gRNAs bearing truncations in their 5′ complementarity regions). Lengths of gRNA complementarity regions and corresponding target DNA sites (SEQ ID NOs: 2708-2721, respectively, in order of appearance) are shown. Indel frequencies were measured by T7EI assay. Ctrl=control gRNA lacking a complementarity region.

FIG. 3C: DNA sequences of indel mutations (SEQ ID NOs: 2722-2754, respectively, in order of appearance) induced by RGNs using a tru-gRNA or a matched full-length gRNA targeted to the EMX1 site. The portion of the target DNA site that interacts with the gRNA complementarity region is highlighted in grey with the first base of the PAM sequence shown in lowercase. Deletions are indicated by dashes highlighted in grey and insertions by italicized letters highlighted in grey. The net number of bases deleted or inserted and the number of times each sequence was isolated are shown to the right.

FIG. 3D: Efficiencies of precise HDR/ssODN-mediated alterations introduced at two endogenous human genes by matched standard and tru-RGNs. % HDR was measured using a BamHI restriction digest assay (see the Experimental Procedures for Example 2). Control gRNA=empty U6 promoter vector.

FIG. 3E: U2OS.EGFP cells were transfected with variable amounts of full-length gRNA expression plasmids (top) or tru-gRNA expression plasmids (bottom) together with a fixed amount of Cas9 expression plasmid and then assayed for percentage of cells with decreased EGFP expression. Mean values from duplicate experiments are shown with standard errors of the mean. Note that the data obtained with tru-gRNA matches closely with data from experiments performed with full-length gRNA expression plasmids instead of tru-gRNA plasmids for these three EGFP target sites.

FIG. 3F: U2OS.EGFP cells were transfected with variable amount of Cas9 expression plasmid together with fixed amounts of full-length gRNA expression plasmids (top) or tru-gRNA expression plasmids (bottom) for each target (amounts determined for each tru-gRNA from the experiments of FIG. 3E). Mean values from duplicate experiments are shown with standard errors of the mean. Note that the data obtained with tru-gRNA matches closely with data from experiments performed with full-length gRNA expression plasmids instead of tru-gRNA plasmids for these three EGFP target sites. The results of these titrations determined the concentrations of plasmids used in the EGFP disruption assays performed in Examples 1 and 2.

FIG. 4A: Schematic illustrating locations of VEGFA sites 1 and 4 (SEQ ID NOs: 2755-2756, respectively, in order of appearance) targeted by gRNAs for paired double nicks. Target sites for the full-length gRNAs are underlined with the first base in the PAM sequence shown in lowercase. Location of the BamHI restriction site inserted by HDR with a ssODN donor is shown.

FIG. 4B: A tru-gRNA can be used with a paired nickase strategy to efficiently induce indel mutations. Substitution of a full-length gRNA for VEGFA site 1 with a tru-gRNA does not reduce the efficiency of indel mutations observed with a paired full-length gRNA for VEGFA site 4 and Cas9-D10A nickases. Control gRNA used is one lacking a complementarity region.

FIG. 4C: A tru-gRNA can be used with a paired nickase strategy to efficiently induce precise HDR/ssODN-mediated sequence alterations. Substitution of a full-length gRNA for VEGFA site 1 with a tru-gRNA does not reduce the efficiency of indel mutations observed with a paired full-length gRNA for VEGFA site 4 and Cas9-D10A nickases with an ssODN donor template. Control gRNA used is one lacking a complementarity region.

FIG. 5A: Activities of RGNs targeted to three sites in EGFP using full-length (top) or tru-gRNAs (bottom) with single mismatches at each position (except at the 5′-most base which must remain a G for efficient expression from the U6 promoter). Grey boxes in the grid below represent positions of the Watson-Crick transversion mismatches. Empty gRNA control used is a gRNA lacking a complementarity region. RGN activities were measured using the EGFP disruption assay and values shown represent the percentage of EGFP-negative observed relative to an RGN using a perfectly matched gRNA. Experiments were performed in duplicate and means with error bars representing standard errors of the mean are shown.

FIG. 5B: Activities of RGNs targeted to three sites in EGFP using full-length (top) or tru-gRNAs (bottom) with adjacent double mismatches at each position (except at the 5′-most base which must remain a G for efficient expression from the U6 promoter). Data presented as in 5A.

FIG. 6A: Absolute frequencies of on- and off-target indel mutations induced by RGNs targeted to three different endogenous human gene sites as measured by deep sequencing. Indel frequencies are shown for the three target sites from cells in which targeted RGNs with a full-length gRNA, a tru-gRNA, or a control gRNA lacking a complementarity region were expressed. Absolute counts of indel mutations used to make these graphs can be found in Table 3B.

FIG. 6B: Fold-improvements in off-target site specificities of three tru-RGNs. Values shown represent the ratio of on/off-target activities of tru-RGNs to on/off-target activities of standard RGNs for the off-target sites shown, calculated using the data from (A) and Table 3B. For the sites marked with an asterisk (*), no indels were observed with the tru-RGN and therefore the values shown represent conservative statistical estimates for the fold-improvements in specificities for these off-target sites (see Results and Experimental Procedures).

FIG. 6C, top: Comparison of the on-target and an off-target site (SEQ ID NOs: 2757 and 2758, respectively, in order of appearance) identified by T7EI assay for the tru-RGN targeted to VEGFA site 1 (more were identified by deep sequencing). Note that the full-length gRNA is mismatched to the two nucleotides at the 5′ end of the target site and that these are the two nucleotides not present in the tru-gRNA target site. Mismatches in the off-target site relative to the on-target are highlighted in bold underlined text. Mismatches between the gRNAs and the off-target site are shown with X's.

FIG. 6C, bottom: Indel mutation frequencies induced in the off-target site by RGNs bearing full-length or truncated gRNAs (SEQ ID NOs: 2759 and 2760, respectively, in order of appearance). Indel mutation frequencies were determined by T7EI assay. Note that the off-target site in this figure is one that we had examined previously for indel mutations induced by the standard RGN targeted to VEGFA site 1 and designated as site OT1-30 in that earlier study (Example 1 and Fu et al., Nat Biotechnol. 31(9):822-6 (2013)). It is likely that we did not identify off target mutations at this site in our previous experiments because the frequency of indel mutations appears to be at the reliable detection limit of the T7EI assay (2-5%).

FIGS. 7A-D: DNA sequences of indel mutations (SEQ ID NOs: 2761-2888, respectively, in order of appearance) induced by RGNs using tru-gRNAs or matched full-length gRNAs targeted to VEGFA sites 1 and 3. Sequences depicted as in FIG. 3C.

FIG. 7E. Indel mutation frequencies induced by tru-gRNAs bearing a mismatched 5′ G nucleotide. Indel mutation frequencies in human U2OS.EGFP cells induced by Cas9 directed by tru-gRNAs bearing 17, 18 or 20 nt complementarity regions for VEGFA sites 1 and 3 and EMX1 site 1 (SEQ ID NOs: 2890-2898, respectively, in order of appearance) are shown. Three of these gRNAs contain a mismatched 5′ G (indicated by positions marked in bold text). Bars indicate results from experiments using full-length gRNA (20 nt), tru-gRNA (17 or 18 nt), and tru-gRNA with a mismatched 5′ G nucleotide (17 or 18 nt with boldface T at 5′ end). (Note that no activity was detectable for the mismatched tru-gRNA to EMX1 site 1.)

FIGS. 8A-C: Sequences of off-target indel mutations (SEQ ID NOs: 2899-2974, respectively, in order of appearance) induced by RGNs in human U2OS.EGFP cells. Wild-type genomic off-target sites recognized by RGNs (including the PAM sequence) are highlighted in grey and numbered as in Table 1 and Table B. Note that the complementary strand is shown for some sites. Deleted bases are shown as dashes on a grey background. Inserted bases are italicized and highlighted in grey.

FIGS. 9A-C: Sequences of off-target indel mutations (SEQ ID NOs: 2975-3037 and 2889, respectively, in order of appearance) induced by RGNs in human HEK293 cells. Wild-type genomic off-target sites recognized by RGNs (including the PAM sequence) are highlighted in grey and numbered as in Table 1 and Table B. Note that the complementary strand is shown for some sites. Deleted bases are shown as dashes on a grey background. Inserted bases are italicized and highlighted in grey. *Yielded a large number of single by indels.

DETAILED DESCRIPTION

CRISPR RNA-guided nucleases (RGNs) have rapidly emerged as a facile and efficient platform for genome editing. Although Marraffini and colleagues (Jiang et al., Nat Biotechnol 31, 233-239 (2013)) recently performed a systematic investigation of Cas9 RGN specificity in bacteria, the specificities of RGNs in human cells have not been extensively defined. Understanding the scope of RGN-mediated off-target effects in human and other eukaryotic cells will be critically essential if these nucleases are to be used widely for research and therapeutic applications. The present inventors have used a human cell-based reporter assay to characterize off-target cleavage of Cas9-based RGNs. Single and double mismatches were tolerated to varying degrees depending on their position along the guide RNA (gRNA)-DNA interface. Off-target alterations induced by four out of six RGNs targeted to endogenous loci in human cells were readily detected by examination of partially mismatched sites. The off-target sites identified harbor up to five mismatches and many are mutagenized with frequencies comparable to (or higher than) those observed at the intended on-target site. Thus RGNs are highly active even with imperfectly matched RNA-DNA interfaces in human cells, a finding that might confound their use in research and therapeutic applications.

The results described herein reveal that predicting the specificity profile of any given RGN is neither simple nor straightforward. The EGFP reporter assay experiments show that single and double mismatches can have variable effects on RGN activity in human cells that do not strictly depend upon their position(s) within the target site. For example, consistent with previously published reports, alterations in the 3′ half of the sgRNA/DNA interface generally have greater effects than those in the 5′ half (Jiang et al., Nat Biotechnol 31, 233-239 (2013); Cong et al., Science 339, 819-823 (2013); Jinek et al., Science 337, 816-821 (2012)); however, single and double mutations in the 3′ end sometimes also appear to be well tolerated whereas double mutations in the 5′ end can greatly diminish activities. In addition, the magnitude of these effects for mismatches at any given position(s) appears to be site-dependent. Comprehensive profiling of a large series of RGNs with testing of all possible nucleotide substitutions (beyond the Watson-Crick transversions used in our EGFP reporter experiments) may help provide additional insights into the range of potential off-targets. In this regard, the recently described bacterial cell-based method of Marraffini and colleagues (Jiang et al., Nat Biotechnol 31, 233-239 (2013)) or the in vitro, combinatorial library-based cleavage site-selection methodologies previously applied to ZFNs by Liu and colleagues (Pattanayak et al., Nat Methods 8, 765-770 (2011)) might be useful for generating larger sets of RGN specificity profiles.

Despite these challenges in comprehensively predicting RGN specificities, it was possible to identify bona fide off-targets of RGNs by examining a subset of genomic sites that differed from the on-target site by one to five mismatches. Notably, under conditions of these experiments, the frequencies of RGN-induced mutations at many of these off-target sites were similar to (or higher than) those observed at the intended on-target site, enabling the detection of mutations at these sites using the T7EI assay (which, as performed in our laboratory, has a reliable detection limit of ˜2 to 5% mutation frequency). Because these mutation rates were very high, it was possible to avoid using deep sequencing methods previously required to detect much lower frequency ZFN- and TALEN-induced off-target alterations (Pattanayak et al., Nat Methods 8, 765-770 (2011); Perez et al., Nat Biotechnol 26, 808-816 (2008); Gabriel et al., Nat Biotechnol 29, 816-823 (2011); Hockemeyer et al., Nat Biotechnol 29, 731-734 (2011)). Analysis of RGN off-target mutagenesis in human cells also confirmed the difficulties of predicting RGN specificities—not all single and double mismatched off-target sites show evidence of mutation whereas some sites with as many as five mismatches can also show alterations. Furthermore, the bona fide off-target sites identified do not exhibit any obvious bias toward transition or transversion differences relative to the intended target sequence (Table E; grey highlighted rows).

Although off-target sites were seen for a number of RGNs, identification of these sites was neither comprehensive nor genome-wide in scale. For the six RGNs studied, only a very small subset of the much larger total number of potential off-target sequences in the human genome (sites that differ by three to six nucleotides from the intended target site; compare Tables E and C) was examined Although examining such large numbers of loci for off-target mutations by T7EI assay is neither a practical nor a cost-effective strategy, the use of high-throughput sequencing in future studies might enable the interrogation of larger numbers of candidate off-target sites and provide a more sensitive method for detecting bona fide off-target mutations. For example, such an approach might enable the unveiling of additional off-target sites for the two RGNs for which we failed to uncover any off-target mutations. In addition, an improved understanding both of RGN specificities and of any epigenomic factors (e.g., DNA methylation and chromatin status) that may influence RGN activities in cells might also reduce the number of potential sites that need to be examined and thereby make genome-wide assessments of RGN off-targets more practical and affordable.

As described herein, a number of strategies can be used to minimize the frequencies of genomic off-target mutations. For example, the specific choice of RGN target site can be optimized; given that off-target sites that differ at up to five positions from the intended target site can be efficiently mutated by RGNs, choosing target sites with minimal numbers of off-target sites as judged by mismatch counting seems unlikely to be effective; thousands of potential off-target sites that differ by four or five positions within the 20 bp RNA:DNA complementarity region will typically exist for any given RGN targeted to a sequence in the human genome (see, for example, Table C). It is also possible that the nucleotide content of the gRNA complementarity region might influence the range of potential off-target effects. For example, high GC-content has been shown to stabilize RNA:DNA hybrids (Sugimoto et al., Biochemistry 34, 11211-11216 (1995)) and therefore might also be expected to make gRNA/genomic DNA hybridization more stable and more tolerant to mismatches. Additional experiments with larger numbers of gRNAs will be needed to assess if and how these two parameters (numbers of mismatched sites in the genome and stability of the RNA:DNA hybrid) influence the genome-wide specificities of RGNs. However, it is important to note that even if such predictive parameters can be defined, the effect of implementing such guidelines would be to further restrict the targeting range of RGNs.

One potential general strategy for reducing RGN-induced off-target effects might be to reduce the concentrations of gRNA and Cas9 nuclease expressed in the cell. This idea was tested using the RGNs for VEGFA target sites 2 and 3 in U2OS.EGFP cells; transfecting less sgRNA- and Cas9-expressing plasmid decreased the mutation rate at the on-target site but did not appreciably change the relative rates of off-target mutations (Tables 2A and 2B). Consistent with this, high-level off-target mutagenesis rates were also observed in two other human cell types (HEK293 and K562 cells) even though the absolute rates of on-target mutagenesis are lower than in U2OS.EGFP cells. Thus, reducing expression levels of gRNA and Cas9 in cells is not likely to provide a solution for reducing off-target effects. Furthermore, these results also suggest that the high rates of off-target mutagenesis observed in human cells are not caused by overexpression of gRNA and/or Cas9.

TABLE 2A Indel mutation frequencies at on- and off-target genomic sites  induced by different amounts of Cas9- and single gRNA-expressing  plasmids for the RGN targeted to VEGFA Target Site 2 250 ng gRNA/ 12.5 ng gRNA/ 750 ng Cas9 50 ng Cas9 SEQ Mean indel Mean indel ID frequency frequency Site Sequence NO: (%) ± SEM (%) ± SEM T2 GACCCCCTCCACCCCGCCTCCGG 12 50.2 ± 4.9 25.4 ± 4.8 (On-target) OT2-1 GACCCCC C CCACCCCGCC C CCGG 13 14.4 ± 3.4  4.2 ± 0.2 OT2-2 G GG CCCCTCCACCCCGCCTCTGG 14 20.0 ± 6.2  9.8 ± 1.1 OT2-6 CTA CCCCTCCACCCCGCCTCCGG 15  8.2 ± 1.4  6.0 ± 0.5 OT2-9 G C CCCC AC CCACCCCGCCTCTGG 16 50.7 ± 5.6 16.4 ± 2.1 OT2-15 T ACCCCC CA CACCCCGCCTCTGG 17  9.7 ± 4.5  2.1 ± 0.0 OT2-17 ACA CCCC C CCACCCCGCCTCAGG 18 14.0 ± 2.8  7.1 ± 0.0 OT2-19 ATT CCCC C CCACCCCGCCTCAGG 19 17.0 ± 3.3  9.2 ± 0.4 OT2-20 CC CC A CC C CCACCCCGCCTCAGG 20  6.1 ± 1.3 N.D. OT2-23 CG CCC T C C CCACCCCGCCTCCGG 21 44.4 ± 6.7 35.1 ± 1.8 OT2-24 CT CCCC AC CCACCCCGCCTCAGG 22 62.8 ± 5.0 44.1 ± 4.5 OT2-29 TG CCCC TC CCACCCCGCCTCTGG 23 13.8 ± 5.2  5.0 ± 0.2 OT2-34 AGG CCCC CA CACCCCGCCTCAGG 24  2.8 ± 1.5 N.D. Amounts of gRNA- and Cas9-expressing plasmids transfected into U2OS.EGFP cells for these assays are shown at the top of each column. (Note that data for 250 ng gRNA/750 ng Cas9 are the same as those presented in Table 1.) Mean indel frequencies were determined using the T7EI assay from replicate samples as described in Methods. OT = Off-target sites, numbered as in Table 1 and Table B. Mismatches from the on-target site (within the 20 bp region to which the gRNA hybridizes) are highlighted as bold, underlined text. N.D. = none detected

TABLE 2B Indel mutation frequencies at on- and off-target genomic sites induced by different amounts of Cas9- and single gRNA-expressing plasmids for the RGN targeted to VEGFA Target Site 3 250 ng gRNA/ 12.5 ng gRNA/ 750 ng Cas9 250 ng Cas9 SEQ Mean indel Mean indel ID frequency frequency Site Sequence NO: (%) ± SEM (%) ± SEM T3 GGTGAGTGAGTGTGTGCGTGTGG 25 49.4 ± 3.8 33.0 ± 3.7 (On-target) OT3-1 GGTGAGTGAGTGTGTG T GTGAGG 26  7.4 ± 3.4 N.D. OT3-2 A GTGAGTGAGTGTGTG T GTGGGG 27 24.3 ± 9.2  9.8 ± 4.2 OT3-4 GCTGAGTGAGTGT A TGCGTGTGG 28 20.9 ± 11.8  4.2 ± 1.2 OT3-9 GGTGAGTGAGTG C GTGCG G GTGG 29  3.2 ± 0.3 N.D. OT3-17 GTTGAGTGA A TGTGTGCGTGAGG 30  2.9 ± 0.2 N.D. OT3-18 T GTG G GTGAGTGTGTGCGTGAGG 31 13.4 ± 4.2  4.9 ± 0.0 OT3-20 A G A GAGTGAGTGTGTGC A TGAGG 32 16.7 ± 3.5  7.9 ± 2.4 Amounts of gRNA- and Cas9-expressing plasmids transfected into U2OS.EGFP cells for these assays are shown at the top of each column. (Note that data for 250 ng gRNA/750 ng Cas9 are the same as those presented in Table 1.) Mean indel frequencies were determined using the T7EI assay from replicate samples as described in Methods. OT = Off-target sites, numbered as in Table 1 and Table B. N.D. = none detected

The finding that significant off-target mutagenesis can be induced by RGNs in three different human cell types has important implications for broader use of this genome-editing platform. For research applications, the potentially confounding effects of high frequency off-target mutations will need to be considered, particularly for experiments involving either cultured cells or organisms with slow generation times for which the outcrossing of undesired alterations would be challenging. One way to control for such effects might be to utilize multiple RGNs targeted to different DNA sequences to induce the same genomic alteration because off-target effects are not random but instead related to the targeted site. However, for therapeutic applications, these findings clearly indicate that the specificities of RGNs will need to be carefully defined and/or improved if these nucleases are to be used safely in the longer term for treatment of human diseases.

Methods for Improving Specificity

As shown herein, CRISPR-Cas RNA-guided nucleases based on the S. pyogenes Cas9 protein can have significant off-target mutagenic effects that are comparable to or higher than the intended on-target activity (Example 1). Such off-target effects can be problematic for research and in particular for potential therapeutic applications. Therefore, methods for improving the specificity of CRISPR-Cas RNA guided nucleases (RGNs) are needed.

As described in Example 1, Cas9 RGNs can induce high-frequency indel mutations at off-target sites in human cells (see also Cradick et al., 2013; Fu et al., 2013; Hsu et al., 2013; Pattanayak et al., 2013). These undesired alterations can occur at genomic sequences that differ by as many as five mismatches from the intended on-target site (see Example 1). In addition, although mismatches at the 5′ end of the gRNA complementarity region are generally better tolerated than those at the 3′ end, these associations are not absolute and show site-to-site-dependence (see Example 1 and Fu et al., 2013; Hsu et al., 2013; Pattanayak et al., 2013). As a result, computational methods that rely on the number and/or positions of mismatches currently have limited predictive value for identifying bona fide off-target sites. Therefore, methods for reducing the frequencies of off-target mutations remain an important priority if RNA-guided nucleases are to be used for research and therapeutic applications.

Truncated Guide RNAs (Tru-gRNAs) Achieve Greater Specificity

Guide RNAs generally speaking come in two different systems: System 1, which uses separate crRNA and tracrRNAs that function together to guide cleavage by Cas9, and System 2, which uses a chimeric crRNA-tracrRNA hybrid that combines the two separate guide RNAs in a single system (referred to as a single guide RNA or sgRNA, see also Jinek et al., Science 2012; 337:816-821). The tracrRNA can be variably truncated and a range of lengths has been shown to function in both the separate system (system 1) and the chimeric gRNA system (system 2). For example, in some embodiments, tracrRNA may be truncated from its 3′ end by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35 or 40 nts. In some embodiments, the tracrRNA molecule may be truncated from its 5′ end by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35 or 40 nts. Alternatively, the tracrRNA molecule may be truncated from both the 5′ and 3′ end, e.g., by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 nts on the 5′ end and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35 or 40 nts on the 3′ end. See, e.g., Jinek et al., Science 2012; 337:816-821; Mali et al., Science. 2013 Feb. 15; 339(6121):823-6; Cong et al., Science. 2013 Feb. 15; 339(6121):819-23; and Hwang and Fu et al., Nat Biotechnol. 2013 March; 31(3):227-9; Jinek et al., Elife 2, e00471 (2013)). For System 2, generally the longer length chimeric gRNAs have shown greater on-target activity but the relative specificities of the various length gRNAs currently remain undefined and therefore it may be desirable in certain instances to use shorter gRNAs. In some embodiments, the gRNAs are complementary to a region that is within about 100-800 bp upstream of the transcription start site, e.g., is within about 500 bp upstream of the transcription start site, includes the transcription start site, or within about 100-800 bp, e.g., within about 500 bp, downstream of the transcription start site. In some embodiments, vectors (e.g., plasmids) encoding more than one gRNA are used, e.g., plasmids encoding, 2, 3, 4, 5, or more gRNAs directed to different sites in the same region of the target gene.

The present application describes a strategy for improving RGN specificity based on the seemingly counterintuitive idea of shortening, rather than lengthening, the gRNA complementarity region. These shorter gRNAs can induce various types of Cas9-mediated on-target genome editing events with efficiencies comparable to (or, in some cases, higher than) full-length gRNAs at multiple sites in a single integrated EGFP reporter gene and in endogenous human genes. In addition, RGNs using these shortened gRNAs exhibit increased sensitivity to small numbers of mismatches at the gRNA-target DNA interface. Most importantly, use of shortened gRNAs substantially reduces the rates of genomic off-target effects in human cells, yielding improvements of specificity as high as 5000-fold or more at these sites. Thus, this shortened gRNA strategy provides a highly effective approach for reducing off-target effects without compromising on-target activity and without the need for expression of a second, potentially mutagenic gRNA. This approach can be implemented on its own or in conjunction with other strategies such as the paired nickase method to reduce the off-target effects of RGNs in human cells.

Thus, one method to enhance specificity of CRISPR/Cas nucleases shortens the length of the guide RNA (gRNA) species used to direct nuclease specificity. Cas9 nuclease can be guided to specific 17-18 nt genomic targets bearing an additional proximal protospacer adjacent motif (PAM), e.g., of sequence NGG, using a guide RNA, e.g., a single gRNA or a crRNA (paired with a tracrRNA), bearing 17 or 18 nts at its 5′ end that are complementary to the complementary strand of the genomic DNA target site (FIG. 1).

Although one might expect that increasing the length of the gRNA complementarity region would improve specificity, the present inventors (Hwang et al., PLoS One. 2013 Jul. 9; 8(7):e68708) and others (Ran et al., Cell. 2013 Sep. 12; 154(6):1380-9) have previously observed that lengthening the target site complementarity region at the 5′ end of the gRNA actually makes it function less efficiently at the on-target site.

By contrast, experiments in Example 1 showed that gRNAs bearing multiple mismatches within a standard length 5′ complementarity targeting region could still induce robust Cas9-mediated cleavage of their target sites. Thus, it was possible that truncated gRNAs lacking these 5′-end nucleotides might show activities comparable to their full-length counterparts (FIG. 2A). It was further speculated that these 5′ nucleotides might normally compensate for mismatches at other positions along the gRNA-target DNA interface and therefore predicted that shorter gRNAs might be more sensitive to mismatches and thus induce lower levels of off-target mutations (FIG. 2A).

Decreasing the length of the DNA sequence targeted might also decrease the stability of the gRNA:DNA hybrid, making it less tolerant of mismatches and thereby making the targeting more specific. That is, truncating the gRNA sequence to recognize a shorter DNA target might actually result in a RNA-guided nuclease that is less tolerant to even single nucleotide mismatches and is therefore more specific and has fewer unintended off-target effects.

This strategy for shortening the gRNA complementarity region could potentially be used with RNA guided proteins other than S. pyogenes Cas9 including other Cas proteins from bacteria or archaea as well as Cas9 variants that nick a single strand of DNA or have no-nuclease activity such as a dCas9 bearing catalytic inactivating mutations in one or both nuclease domains. This strategy can be applied to systems that utilize a single gRNA as well as those that use dual gRNAs (e.g., the crRNA and tracrRNA found in naturally occurring systems).

Thus, described herein is a single guide RNA comprising a crRNA fused to a normally trans-encoded tracrRNA, e.g., a single Cas9 guide RNA as described in Mali et al., Science 2013 Feb. 15; 339(6121):823-6, but with a sequence at the 5′ end that is complementary to fewer than 20 nucleotides (nts), e.g., 19, 18, or 17 nts, preferably 17 or 18 nts, of the complementary strand to a target sequence immediately 5′ of a protospacer adjacent motif (PAM), e.g., NGG, NAG, or NNGG. In some embodiments, the shortened Cas9 guide RNA consists of the sequence:

(SEQ ID NO: 2404) (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUA; (SEQ ID NO: 2407) (X₁₇₋₁₈ or X₁₇₋₁₉) GUUUUAGAGCUAUGCUGUUUUG; or (SEQ ID NO: 2408) (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUAUGCU; (SEQ ID NO: 1) (X₁₇₋₁₈ or X₁₇₋₁₉) GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCG(X_(N)); (SEQ ID NO: 2) (X₁₇₋₁₈ or X₁₇₋₁₉) GUUUUAGAGCUAUGCUGAAAAGCAUAGCAAGUUAAAAUAAGGCU AGUCCGUUAUC(X_(N)); (SEQ ID NO: 3) (X₁₇₋₁₈ or X₁₇₋₁₉) GUUUUAGAGCUAUGCUGUUUUGGAAACAAAACAGCAUAGCAAGU UAAAAUAAGGCUAGUCCGUUAUC(X_(N)); (SEQ ID NO: 4) (X₁₇₋₁₈ or X₁₇₋₁₉) GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUU AUCAACUUGAAAAAGUGGCACCGAGUCGGUGC(X_(N)), (SEQ ID NO: 5) (X₁₇₋₁₈ or X₁₇₋₁₉) GUUUAAGAGCUAGAAAUAGCAAGUUUAAAUAAGGCUAGUCCGUU AUCAACUUGAAAAAGUGGCACCGAGUCGGUGC; (SEQ ID NO: 6) (X₁₇₋₁₈ or X₁₇₋₁₉) GUUUUAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGG CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC; or (SEQ ID NO: 7) (X₁₇₋₁₈ or X₁₇₋₁₉) GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGG CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC; wherein X₁₇₋₁₈ or X₁₇₋₁₉ is the nucleotide sequence complementary to 17-18 or 17-19 consecutive nucleotides of the target sequence, respectively. Also described herein are DNAs encoding the shortened Cas9 guide RNAs that have been described previously in the literature (Jinek et al., Science. 337(6096):816-21 (2012) and Jinek et al., Elife. 2:e00471 (2013)).

The guide RNAs can include X_(N) which can be any sequence, wherein N (in the RNA) can be 0-200, e.g., 0-100, 0-50, or 0-20, that does not interfere with the binding of the ribonucleic acid to Cas9.

In some embodiments, the guide RNA includes one or more Adenine (A) or Uracil (U) nucleotides on the 3′ end. In some embodiments the RNA includes one or more U, e.g., 1 to 8 or more Us (e.g., U, UU, UUU, UUUU, UUUUU, UUUUUU, UUUUUUU, UUUUUUUU) at the 3′ end of the molecule, as a result of the optional presence of one or more Ts used as a termination signal to terminate RNA PolIII transcription.

Modified RNA oligonucleotides such as locked nucleic acids (LNAs) have been demonstrated to increase the specificity of RNA-DNA hybridization by locking the modified oligonucleotides in a more favorable (stable) conformation. For example, 2′-O-methyl RNA is a modified base where there is an additional covalent linkage between the 2′ oxygen and 4′ carbon which when incorporated into oligonucleotides can improve overall thermal stability and selectivity (formula I).

Thus in some embodiments, the tru-gRNAs disclosed herein may comprise one or more modified RNA oligonucleotides. For example, the truncated guide RNAs molecules described herein can have one, some or all of the 17-18 or 17-19 nts 5′ region of the guideRNA complementary to the target sequence are modified, e.g., locked (2′-O-4′-C methylene bridge), 5′-methylcytidine, 2′-O-methyl-pseudouridine, or in which the ribose phosphate backbone has been replaced by a polyamide chain (peptide nucleic acid), e.g., a synthetic ribonucleic acid.

In other embodiments, one, some or all of the nucleotides of the tru-gRNA sequence may be modified, e.g., locked (2′-O-4′-C methylene bridge), 5′-methylcytidine, 2′-O-methyl-pseudouridine, or in which the ribose phosphate backbone has been replaced by a polyamide chain (peptide nucleic acid), e.g., a synthetic ribonucleic acid.

In a cellular context, complexes of Cas9 with these synthetic gRNAs could be used to improve the genome-wide specificity of the CRISPR/Cas9 nuclease system.

Exemplary modified or synthetic tru-gRNAs may comprise, or consist of, the following sequences:

(SEQ ID NO: 2404) (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUA(X_(N)); (SEQ ID NO: 2407) (X₁₇₋₁₈ or X₁₇₋₁₉) GUUUUAGAGCUAUGCUGUUUUG (X_(N)); (SEQ ID NO: 2408) (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUAUGCU(X_(N)); (SEQ ID NO: 1) (X₁₇₋₁₈ or X₁₇₋₁₉) GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCG(X_(N)); (SEQ ID NO: 2) (X₁₇₋₁₈ or X₁₇₋₁₉) GUUUUAGAGCUAUGCUGAAAAGCAUAGCAAGUUAAAAUAAGGCU AGUCCGUUAUC(X_(N)); (SEQ ID NO: 3) (X₁₇₋₁₈ or X₁₇₋₁₉) GUUUUAGAGCUAUGCUGUUUUGGAAACAAAACAGCAUAGCAAGU UAAAAUAAGGCUAGUCCGUUAUC(X_(N)); (SEQ ID NO: 4) (X₁₇₋₁₈) GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUU AUCAACUUGAAAAAGUGGCACCGAGUCGGUGC(X_(N)), (SEQ ID NO: 5) (X₁₇₋₁₈ or X₁₇₋₁₉) GUUUAAGAGCUAGAAAUAGCAAGUUUAAAUAAGGCUAGUCCGUU AUCAACUUGAAAAAGUGGCACCGAGUCGGUGC; (SEQ ID NO: 6) (X₁₇₋₁₈ or X₁₇₋₁₉) GUUUUAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGG CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC; or (SEQ ID NO: 7) (X₁₇₋₁₈ or X₁₇₋₁₉) GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGG CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC; wherein X₁₇₋₁₈ or X₁₇₋₁₉ is a sequence complementary to 17-18 or 17-19 nts of a target sequence, respectively, preferably a target sequence immediately 5′ of a protospacer adjacent motif (PAM), e.g., NGG, NAG, or NNGG, and further wherein one or more of the nucleotides are locked, e.g., one or more of the nucleotides within the sequence X₁₇₋₁₈ or X₁₇₋₁₉, one or more of the nucleotides within the sequence X_(N), or one or more of the nucleotides within any sequence of the tru-gRNA. X_(N) is any sequence, wherein N (in the RNA) can be 0-200, e.g., 0-100, 0-50, or 0-20, that does not interfere with the binding of the ribonucleic acid to Cas9. In some embodiments the RNA includes one or more U, e.g., 1 to 8 or more Us (e.g., U, UU, UUU, UUUU, UUUUU, UUUUUU, UUUUUUU, UUUUUUUU) at the 3′ end of the molecule, as a result of the optional presence of one or more Ts used as a termination signal to terminate RNA PolIII transcription. Although some of the examples described herein utilize a single gRNA, the methods can also be used with dual gRNAs (e.g., the crRNA and tracrRNA found in naturally occurring systems). In this case, a single tracrRNA would be used in conjunction with multiple different crRNAs expressed using the present system, e.g., the following: (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUA (SEQ ID NO:2404); (X₁₇₋₁₈ or X₁₇₋₁₉) GUUUUAGAGCUAUGCUGUUUUG (SEQ ID NO:2407); or (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUAUGCU (SEQ ID NO:2408); and a tracrRNA sequence. In this case, the crRNA is used as the guide RNA in the methods and molecules described herein, and the tracrRNA can be expressed from the same or a different DNA molecule. In some embodiments, the methods include contacting the cell with a tracrRNA comprising or consisting of the sequence GGAACCAUUCAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUA UCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO:8) or an active portion thereof (an active portion is one that retains the ability to form complexes with Cas9 or dCas9). In some embodiments, the tracrRNA molecule may be truncated from its 3′ end by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35 or 40 nts. In another embodiment, the tracrRNA molecule may be truncated from its 5′ end by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35 or 40 nts. Alternatively, the tracrRNA molecule may be truncated from both the 5′ and 3′ end, e.g., by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 nts on the 5′ end and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35 or 40 nts on the 3′ end. Exemplary tracrRNA sequences in addition to SEQ ID NO:8 include the following: UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGC (SEQ ID NO:2405) or an active portion thereof; AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGU GGCACCGAGUCGGUGC (SEQ ID NO:2407) or an active portion thereof; CAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGA AAAAGUGGCACCGAGUCGGUGC (SEQ ID NO:2409) or an active portion thereof; UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG (SEQ ID NO:2410) or an active portion thereof; UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCA (SEQ ID NO:2411) or an active portion thereof; or UAGCAAGUUAAAAUAAGGCUAGUCCG (SEQ ID NO:2412) or an active portion thereof.

In some embodiments wherein (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUAUGCUGUUUUG (SEQ ID NO:2407) is used as a crRNA, the following tracrRNA is used:

GGAACCAUUCAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUA UCAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO:8) or an active portion thereof. In some embodiments wherein (X₁₇₋₁₈ or X₁₇₋₁₉)GUUUUAGAGCUA (SEQ ID NO:2404) is used as a crRNA, the following tracrRNA is used:

UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGC (SEQ ID NO:2405) or an active portion thereof. In some embodiments wherein (X₁₇₋₁₈ or X₁₇₋₁₉) GUUUUAGAGCUAUGCU (SEQ ID NO:2408) is used as a crRNA, the following tracrRNA is used: AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGU GGCACCGAGUCGGUGC (SEQ ID NO:2406) or an active portion thereof

In addition, in a system that uses separate crRNA and tracrRNA, one or both can be synthetic and include one or more modified (e.g., locked) nucleotides or deoxyribonucleotides.

In some embodiments, the single guide RNAs and/or crRNAs and/or tracrRNAs can include one or more Adenine (A) or Uracil (U) nucleotides on the 3′ end.

Existing Cas9-based RGNs use gRNA-DNA heteroduplex formation to guide targeting to genomic sites of interest. However, RNA-DNA heteroduplexes can form a more promiscuous range of structures than their DNA-DNA counterparts. In effect, DNA-DNA duplexes are more sensitive to mismatches, suggesting that a DNA-guided nuclease may not bind as readily to off-target sequences, making them comparatively more specific than RNA-guided nucleases. Thus, the truncated guide RNAs described herein can be hybrids, i.e., wherein one or more deoxyribonucleotides, e.g., a short DNA oligonucleotide, replaces all or part of the gRNA, e.g., all or part of the complementarity region of a gRNA. This DNA-based molecule could replace either all or part of the gRNA in a single gRNA system or alternatively might replace all of part of the crRNA in a dual crRNA/tracrRNA system. Such a system that incorporates DNA into the complementarity region should more reliably target the intended genomic DNA sequences due to the general intolerance of DNA-DNA duplexes to mismatching compared to RNA-DNA duplexes. Methods for making such duplexes are known in the art, See, e.g., Barker et al., BMC Genomics. 2005 Apr. 22; 6:57; and Sugimoto et al., Biochemistry. 2000 Sep. 19; 39(37):11270-81.

Exemplary modified or synthetic tru-gRNAs may comprise, or consist of, the following sequences:

(SEQ ID NO: 1) (X₁₇₋₁₈ or X₁₇₋₁₉) GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCG(X_(N)); (SEQ ID NO: 2) (X₁₇₋₁₈ or X₁₇₋₁₉) GUUUUAGAGCUAUGCUGAAAAGCAUAGCAAGUUAAAAUAAGGCU AGUCCGUUAUC(X_(N)); (SEQ ID NO: 3) (X₁₇₋₁₈ or X₁₇₋₁₉) GUUUUAGAGCUAUGCUGUUUUGGAAACAAAACAGCAUAGCAAGU UAAAAUAAGGCUAGUCCGUUAUC(X_(N)); (SEQ ID NO: 4) (X₁₇₋₁₈ or X₁₇₋₁₉) GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUU AUCAACUUGAAAAAGUGGCACCGAGUCGGUGC(X_(N)), (SEQ ID NO: 5) (X₁₇₋₁₈ or X₁₇₋₁₉) GUUUAAGAGCUAGAAAUAGCAAGUUUAAAUAAGGCUAGUCCGUU AUCAACUUGAAAAAGUGGCACCGAGUCGGUGC; (SEQ ID NO: 6) (X₁₇₋₁₈ or X₁₇₋₁₉) GUUUUAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGG CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC; or (SEQ ID NO: 7) (X₁₇₋₁₈ or X₁₇₋₁₉) GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGG CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC; wherein X₁₇₋₁₈ or X₁₇₋₁₉ is a sequence complementary to 17-18 or 17-19 nts of a target sequence, respectively, preferably a target sequence immediately 5′ of a protospacer adjacent motif (PAM), e.g., NGG, NAG, or NNGG, and further wherein one or more of the nucleotides are deoxyribonucleotides, e.g., one or more of the nucleotides within the sequence X₁₇₋₁₈ or X₁₇₋₁₉, one or more of the nucleotides within the sequence X_(N), or one or more of the nucleotides within any sequence of the tru-gRNA. X_(N) is any sequence, wherein N (in the RNA) can be 0-200, e.g., 0-100, 0-50, or 0-20, that does not interfere with the binding of the ribonucleic acid to Cas9. In some embodiments the RNA includes one or more U, e.g., 1 to 8 or more Us (e.g., U, UU, UUU, UUUU, UUUUU, UUUUUU, UUUUUUU, UUUUUUUU) at the 3′ end of the molecule, as a result of the optional presence of one or more Ts used as a termination signal to terminate RNA PolIII transcription.

In addition, in a system that uses separate crRNA and tracrRNA, one or both can be synthetic and include one or more deoxyribonucleotides.

In some embodiments, the single guide RNAs or crRNAs or tracrRNAs includes one or more Adenine (A) or Uracil (U) nucleotides on the 3′ end.

In some embodiments, the gRNA is targeted to a site that is at least three or more mismatches different from any sequence in the rest of the genome in order to minimize off-target effects.

The methods described can include expressing in a cell, or contacting the cell with, a shortened Cas9 gRNA (tru-gRNA) as described herein (optionally a modified or DNA/RNA hybrid tru-gRNA), plus a nuclease that can be guided by the shortened Cas9 gRNAs, e.g., a Cas9 nuclease, e.g., as described in Mali et al., a Cas9 nickase as described in Jinek et al., 2012; or a dCas9-heterofunctional domain fusion (dCas9-HFD).

Cas9

A number of bacteria express Cas9 protein variants. The Cas9 from Streptococcus pyogenes is presently the most commonly used; some of the other Cas9 proteins have high levels of sequence identity with the S. pyogenes Cas9 and use the same guide RNAs. Others are more diverse, use different gRNAs, and recognize different PAM sequences as well (the 2-5 nucleotide sequence specified by the protein which is adjacent to the sequence specified by the RNA). Chylinski et al. classified Cas9 proteins from a large group of bacteria (RNA Biology 10:5, 1-12; 2013), and a large number of Cas9 proteins are listed in supplementary FIG. 1 and supplementary table 1 thereof, which are incorporated by reference herein. Additional Cas9 proteins are described in Esvelt et al., Nat Methods. 2013 November; 10(11):1116-21 and Fonfara et al., “Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems.” Nucleic Acids Res. 2013 Nov. 22. [Epub ahead of print] doi:10.1093/nar/gkt1074.

Cas9 molecules of a variety of species can be used in the methods and compositions described herein. While the S. pyogenes and S. thermophilus Cas9 molecules are the subject of much of the disclosure herein, Cas9 molecules of, derived from, or based on the Cas9 proteins of other species listed herein can be used as well. In other words, while the much of the description herein uses S. pyogenes and S. thermophilus Cas9 molecules, Cas9 molecules from the other species can replace them. Such species include those set forth in the following table, which was created based on supplementary FIG. 1 of Chylinski et al., 2013.

Alternative Cas9 proteins GenBank Acc No. Bacterium 303229466 Veillonella atypica ACS-134-V-Col7a 34762592 Fusobacterium nucleatum subsp.vincentii 374307738 Filifactor alocis ATCC 35896 320528778 Solobacterium moorei F0204 291520705 Coprococcus catus GD-7 42525843 Treponema denticola ATCC 35405 304438954 Peptoniphilus duerdenii ATCC BAA-1640 224543312 Catenibacterium mitsuokai DSM 15897 24379809 Streptococcus mutans UA159 15675041 Streptococcus pyogenes SF370 16801805 Listeria innocua Clip11262 116628213 Streptococcus thermophilus LMD-9 323463801 Staphylococcus pseudintermedius ED99 352684361 Acidaminococcus intestini RyC-MR95 302336020 Olsenella uli DSM 7084 366983953 Oenococcus kitaharae DSM 17330 310286728 Bifidobacterium bifidum S17 258509199 Lactobacillus rhamnosus GG 300361537 Lactobacillus gasseri JV-V03 169823755 Finegoldia magna ATCC 29328 47458868 Mycoplasma mobile 163K 284931710 Mycoplasma gallisepticum str. F 363542550 Mycoplasma ovipneumoniae SC01 384393286 Mycoplasma canis PG 14 71894592 Mycoplasma synoviae 53 238924075 Eubacterium rectale ATCC 33656 116627542 Streptococcus thermophilus LMD-9 315149830 Enterococcus faecalis TX0012 315659848 Staphylococcus lugdunensis M23590 160915782 Eubacterium dolichum DSM 3991 336393381 Lactobacillus coryniformis subsp. torquens 310780384 Ilyobacter polytropus DSM 2926 325677756 Ruminococcus albus 8 187736489 Akkermansia muciniphila ATCC BAA-835 117929158 Acidothermus cellulolyticus 11B 189440764 Bifidobacterium longum DJO10A 283456135 Bifidobacterium dentium Bd1 38232678 Corynebacterium diphtheriae NCTC 13129 187250660 Elusimicrobium minutum Pei 191 319957206 Nitratifractor salsuginis DSM 16511 325972003 Sphaerochaeta globus str. Buddy 261414553 Fibrobacter succinogenes subsp. succinogenes 60683389 Bacteroides fragilis NCTC 9343 256819408 Capnocytophaga ochracea DSM 7271 90425961 Rhodopseudomonas palustris BisB18 373501184 Prevotella micans F0438 294674019 Prevotella ruminicola 23 365959402 Flavobacterium columnare ATCC 49512 312879015 Aminomonas paucivorans DSM 12260 83591793 Rhodospirillum rubrum ATCC 11170 294086111 Candidatus Puniceispirillum marinum IMCC1322 121608211 Verminephrobacter eiseniae EF01-2 344171927 Ralstonia syzygii R24 159042956 Dinoroseobacter shibae DFL 12 288957741 Azospirillum sp- B510 92109262 Nitrobacter hamburgensis X14 148255343 Bradyrhizobium sp- BTAil 34557790 Wolinella succinogenes DSM 1740 218563121 Campylobacter jejuni subsp. jejuni 291276265 Helicobacter mustelae 12198 229113166 Bacillus cereus Rock 1-15 222109285 Acidovorax ebreus TPSY 189485225 uncultured Termite group 1 182624245 Clostridium perfringens D str. 220930482 Clostridium cellulolyticum H10 154250555 Parvibaculum lavamentivorans DS-1 257413184 Roseburia intestinalis L1-82 218767588 Neisseria meningitidis Z2491 15602992 Pasteurella multocida subsp. multocida 319941583 Sutterella wadsworthensis 31 254447899 gamma proteobacterium HTCC5015 54296138 Legionella pneumophila str. Paris 331001027 Parasutterella excrementihominis YIT 11859 34557932 Wolinella succinogenes DSM 1740 118497352 Francisella novicida U112 The constructs and methods described herein can include the use of any of those Cas9 proteins, and their corresponding guide RNAs or other guide RNAs that are compatible. The Cas9 from Streptococcus thermophilus LMD-9 CRISPR1 system has also been shown to function in human cells in Cong et al (Science 339, 819 (2013)). Cas9 orthologs from N. meningitides are described in Hou et al., Proc Natl Acad Sci USA. 2013 Sep. 24; 110(39):15644-9 and Esvelt et al., Nat Methods. 2013 November; 10(11):1116-21. Additionally, Jinek et al. showed in vitro that Cas9 orthologs from S. thermophilus and L. innocua, (but not from N. meningitidis or C. jejuni, which likely use a different guide RNA), can be guided by a dual S. pyogenes gRNA to cleave target plasmid DNA, albeit with slightly decreased efficiency.

In some embodiments, the present system utilizes the Cas9 protein from S. pyogenes, either as encoded in bacteria or codon-optimized for expression in mammalian cells, containing mutations at D10, E762, H983, or D986 and H840 or N863, e.g., D10A/D10N and H840A/H840N/H840Y, to render the nuclease portion of the protein catalytically inactive; substitutions at these positions could be alanine (as they are in Nishimasu al., Cell 156, 935-949 (2014)) or they could be other residues, e.g., glutamine, asparagine, tyrosine, serine, or aspartate, e.g., E762Q, H983N, H983Y, D986N, N863D, N863S, or N863H (FIG. 1C). The sequence of the catalytically inactive S. pyogenes Cas9 that can be used in the methods and compositions described herein is as follows; the exemplary mutations of D10A and H840A are in bold and underlined.

(SEQ ID NO: 33)         10         20         30         40         50         60 MDKKYSIGL A  IGTNSVGWAV ITDEYKVPSK KFKVLGNTDR HSIKKNLIGA LLFDSGETAE         70         80         90        100        110        120 ATRLKRTARR RYTRRKNRIC YLQEIFSNEM AKVDDSFFHR LEESFLVEED KKHERHPIFG        130        140        150        160        170        180 NIVDEVAYHE KYPTIYHLRK KLVDSTDKAD LRLIYLALAH MIKFRGHFLI EGDLNPDNSD        190        200        210        220        230        240 VDKLFIQLVQ TYNQLFEENP INASGVDAKA ILSARLSKSR RLENLIAQLP GEKKNGLFGN        250        260        270        280        290        300 LIALSLGLTP NFKSNFDLAE DAKLQLSKDT YDDDLDNLLA QIGDQYADLF LAAKNLSDAI        310        320        330        340        350        360 LLSDILRVNT EITKAPLSAS MIKRYDEHHQ DLTLLKALVR QQLPEKYKEI FFDQSKNGYA        370        380        390        400        410        420 GYIDGGASQE EFYKFIKPIL EKMDGTEELL VKLNREDLLR KQRTFDNGSI PHQIHLGELH        430        440        450        460        470        480 AILRRQEDFY PFLKDNREKI EKILTFRIPY YVGPLARGNS RFAWMTRKSE ETITPWNFEE        490        500        510        520        530        540 VVDKGASAQS FIERMTNFDK NLPNEKVLPK HSLLYEYFTV YNELTKVKYV TEGMRKPAFL        550        560        570        580        590        600 SGEQKKAIVD LLFKTNRKVT VKQLKEDYFK KIECFDSVEI SGVEDRFNAS LGTYHDLLKI        610        620        630        640        650        660 IKDKDFLDNE ENEDILEDIV LTLTLFEDRE MIEERLKTYA HLFDDKVMKQ LKRRRYTGWG        670        680        690        700        710        720 RLSRKLINGI RDKQSGKTIL DFLKSDGFAN RNFMQLIHDD SLTFKEDIQK AQVSGQGDSL        730        740        750        760        770        780 HEHIANLAGS PAIKKGILQT VKVVDELVKV MGRHKPENIV IEMARENQTT QKGQKNSRER        790        800        810        820        830        840 MKRIEEGIKE LGSQILKEHP VENTQLQNEK LYLYYLQNGR DMYVDQELDI NRLSDYDVD A        850        860        870        880        890        900 IVPQSFLKDD SIDNKVLTRS DKNRGKSDNV PSEEVVKKMK NYWRQLLNAK LITQRKFDNL        910        920        930        940        950        960 TKAERGGLSE LDKAGFIKRQ LVETRQITKH VAQILDSRMN TKYDENDKLI REVKVITLKS        970        980        990       1000       1010       1020 KLVSDFRKDF QFYKVREINN YHHAHDAYLN AVVGTALIKK YPKLESEFVY GDYKVYDVRK       1030       1040       1050       1060       1070       1080 MIAKSEQEIG KATAKYFFYS NIMNFFKTEI TLANGEIRKR PLIETNGETG EIVWDKGRDF       1090       1100       1110       1120       1130       1140 ATVRKVLSMP QVNIVKKTEV QTGGFSKESI LPKRNSDKLI ARKKDWDPKK YGGFDSPTVA       1150       1160       1170       1180       1190       1200 YSVLVVAKVE KGKSKKLKSV KELLGITIME RSSFEKNPID FLEAKGYKEV KKDLIIKLPK       1210       1220       1230       1240       1250       1260 YSLFELENGR KRMLASAGEL QKGNELALPS KYVNFLYLAS HYEKLKGSPE DNEQKQLFVE       1270       1280       1290       1300       1310       1320 QHKHYLDEII EQISEFSKRV ILADANLDKV LSAYNKHRDK PIREQAENII HLFTLTNLGA       1330       1340       1350       1360 PAAFKYFDTT IDRKRYTSTK EVLDATLIHQ SITGLYETRI DLSQLGGD

In some embodiments, the Cas9 nuclease used herein is at least about 50% identical to the sequence of S. pyogenes Cas9, i.e., at least 50% identical to SEQ ID NO:33. In some embodiments, the nucleotide sequences are about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO:33. In some embodiments, any differences from SEQ ID NO:33 are in non-conserved regions, as identified by sequence alignment of sequences set forth in Chylinski et al., RNA Biology 10:5, 1-12; 2013 (e.g., in supplementary FIG. 1 and supplementary table 1 thereof); Esvelt et al., Nat Methods. 2013 November; 10(11): 1116-21 and Fonfara et al., Nucl. Acids Res. (2014) 42 (4): 2577-2590. [Epub ahead of print 2013 Nov. 22] doi:10.1093/nar/gkt1074.

To determine the percent identity of two sequences, the sequences are aligned for optimal comparison purposes (gaps are introduced in one or both of a first and a second amino acid or nucleic acid sequence as required for optimal alignment, and non-homologous sequences can be disregarded for comparison purposes). The length of a reference sequence aligned for comparison purposes is at least 50% (in some embodiments, about 50%, 55%, 60%, 65%, 70%, 75%, 85%, 90%, 95%, or 100% of the length of the reference sequence is aligned). The nucleotides or residues at corresponding positions are then compared. When a position in the first sequence is occupied by the same nucleotide or residue as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For purposes of the present application, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package, using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

Cas9-HFD

Cas9-HFD are described in a U.S. Provisional Patent Applications U.S. Ser. No. 61/799,647, Filed on Mar. 15, 2013, U.S. Ser. No. 61/838,148, filed on Jun. 21, 2013, and PCT International Application No. PCT/US14/27335, all of which are incorporated herein by reference in its entirety.

The Cas9-HFD are created by fusing a heterologous functional domain (e.g., a transcriptional activation domain, e.g., from VP64 or NF-κB p65), to the N-terminus or C-terminus of a catalytically inactive Cas9 protein (dCas9). In the present case, as noted above, the dCas9 can be from any species but is preferably from S. pyogenes, In some embodiments, the Cas9 contains mutations in the D10 and H840 residues, e.g., D10N/D10A and H840A/H840N/H840Y, to render the nuclease portion of the protein catalytically inactive, e.g., as shown in SEQ ID NO:33 above.

The transcriptional activation domains can be fused on the N or C terminus of the Cas9. In addition, although the present description exemplifies transcriptional activation domains, other heterologous functional domains (e.g., transcriptional repressors (e.g., KRAB, ERD, SID, and others, e.g., amino acids 473-530 of the ets2 repressor factor (ERF) repressor domain (ERD), amino acids 1-97 of the KRAB domain of KOX1, or amino acids 1-36 of the Mad mSIN3 interaction domain (SID); see Beerli et al., PNAS USA 95:14628-14633 (1998)) or silencers such as Heterochromatin Protein 1 (HP1, also known as swi6), e.g., HP1α or HP1β; proteins or peptides that could recruit long non-coding RNAs (lncRNAs) fused to a fixed RNA binding sequence such as those bound by the MS2 coat protein, endoribonuclease Csy4, or the lambda N protein; enzymes that modify the methylation state of DNA (e.g., DNA methyltransferase (DNMT) or TET proteins); or enzymes that modify histone subunits (e.g., histone acetyltransferases (HAT), histone deacetylases (HDAC), histone methyltransferases (e.g., for methylation of lysine or arginine residues) or histone demethylases (e.g., for demethylation of lysine or arginine residues)) as are known in the art can also be used. A number of sequences for such domains are known in the art, e.g., a domain that catalyzes hydroxylation of methylated cytosines in DNA. Exemplary proteins include the Ten-Eleven-Translocation (TET)1-3 family, enzymes that converts 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) in DNA.

Sequences for human TET1-3 are known in the art and are shown in the following table:

GenBank Accession Nos. Gene Amino Acid Nucleic Acid TET1 NP_085128.2 NM_030625.2 TET2* NP_001120680.1 (var 1) NM_001127208.2 NP_060098.3 (var 2) NM_017628.4 TET3 NP_659430.1 NM_144993.1 *Variant (1) represents the longer transcript and encodes the longer isoform (a). Variant (2) differs in the 5′ UTR and in the 3′ UTR and coding sequence compared to variant 1. The resulting isoform (b) is shorter and has a distinct C-terminus compared to isoform a.

In some embodiments, all or part of the full-length sequence of the catalytic domain can be included, e.g., a catalytic module comprising the cysteine-rich extension and the 2OGFeDO domain encoded by 7 highly conserved exons, e.g., the Tet1 catalytic domain comprising amino acids 1580-2052, Tet2 comprising amino acids 1290-1905 and Tet3 comprising amino acids 966-1678. See, e.g., FIG. 1 of Iyer et al., Cell Cycle. 2009 Jun. 1; 8(11):1698-710. Epub 2009 Jun. 27, for an alignment illustrating the key catalytic residues in all three Tet proteins, and the supplementary materials thereof (available at ftp site ftp.ncbi.nih.gov/pub/aravind/DONS/supplementary_material_DONS.html) for full length sequences (see, e.g., seq 2c); in some embodiments, the sequence includes amino acids 1418-2136 of Tet1 or the corresponding region in Tet2/3.

Other catalytic modules can be from the proteins identified in Iyer et al., 2009.

In some embodiments, the heterologous functional domain is a biological tether, and comprises all or part of (e.g., DNA binding domain from) the MS2 coat protein, endoribonuclease Csy4, or the lambda N protein. These proteins can be used to recruit RNA molecules containing a specific stem-loop structure to a locale specified by the dCas9 gRNA targeting sequences. For example, a dCas9 fused to MS2 coat protein, endoribonuclease Csy4, or lambda N can be used to recruit a long non-coding RNA (lncRNA) such as XIST or HOTAIR; see, e.g., Keryer-Bibens et al., Biol. Cell 100:125-138 (2008), that is linked to the Csy4, MS2 or lambda N binding sequence. Alternatively, the Csy4, MS2 or lambda N protein binding sequence can be linked to another protein, e.g., as described in Keryer-Bibens et al., supra, and the protein can be targeted to the dCas9 binding site using the methods and compositions described herein. In some embodiments, the Csy4 is catalytically inactive.

In some embodiments, the fusion proteins include a linker between the dCas9 and the heterologous functional domains. Linkers that can be used in these fusion proteins (or between fusion proteins in a concatenated structure) can include any sequence that does not interfere with the function of the fusion proteins. In preferred embodiments, the linkers are short, e.g., 2-20 amino acids, and are typically flexible (i.e., comprising amino acids with a high degree of freedom such as glycine, alanine, and serine). In some embodiments, the linker comprises one or more units consisting of GGGS (SEQ ID NO:34) or GGGGS (SEQ ID NO:35), e.g., two, three, four, or more repeats of the GGGS (SEQ ID NO:34) or GGGGS (SEQ ID NO:35) unit. Other linker sequences can also be used.

Expression Systems

In order to use the guide RNAs described, it may be desirable to express them from a nucleic acid that encodes them. This can be performed in a variety of ways. For example, the nucleic acid encoding the guide RNA can be cloned into an intermediate vector for transformation into prokaryotic or eukaryotic cells for replication and/or expression. Intermediate vectors are typically prokaryote vectors, e.g., plasmids, or shuttle vectors, or insect vectors, for storage or manipulation of the nucleic acid encoding the guide RNA for production of the guide RNA. The nucleic acid encoding the guide RNA can also be cloned into an expression vector, for administration to a plant cell, animal cell, preferably a mammalian cell or a human cell, fungal cell, bacterial cell, or protozoan cell.

To obtain expression, a sequence encoding a guide RNA is typically subcloned into an expression vector that contains a promoter to direct transcription. Suitable bacterial and eukaryotic promoters are well known in the art and described, e.g., in Sambrook et al., Molecular Cloning, A Laboratory Manual (3d ed. 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 2010). Bacterial expression systems for expressing the engineered protein are available in, e.g., E. coli, Bacillus sp., and Salmonella (Palva et al., 1983, Gene 22:229-235). Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available.

The promoter used to direct expression of a nucleic acid depends on the particular application. For example, a strong constitutive promoter is typically used for expression and purification of fusion proteins. In contrast, when the guide RNA is to be administered in vivo for gene regulation, either a constitutive or an inducible promoter can be used, depending on the particular use of the guide RNA. In addition, a preferred promoter for administration of the guide RNA can be a weak promoter, such as HSV TK or a promoter having similar activity. The promoter can also include elements that are responsive to transactivation, e.g., hypoxia response elements, Gal4 response elements, lac repressor response element, and small molecule control systems such as tetracycline-regulated systems and the RU-486 system (see, e.g., Gossen & Bujard, 1992, Proc. Natl. Acad. Sci. USA, 89:5547; Oligino et al., 1998, Gene Ther., 5:491-496; Wang et al., 1997, Gene Ther., 4:432-441; Neering et al., 1996, Blood, 88:1147-55; and Rendahl et al., 1998, Nat. Biotechnol., 16:757-761).

In addition to the promoter, the expression vector typically contains a transcription unit or expression cassette that contains all the additional elements required for the expression of the nucleic acid in host cells, either prokaryotic or eukaryotic. A typical expression cassette thus contains a promoter operably linked, e.g., to the nucleic acid sequence encoding the gRNA, and any signals required, e.g., for efficient polyadenylation of the transcript, transcriptional termination, ribosome binding sites, or translation termination. Additional elements of the cassette may include, e.g., enhancers, and heterologous spliced intronic signals.

The particular expression vector used to transport the genetic information into the cell is selected with regard to the intended use of the gRNA, e.g., expression in plants, animals, bacteria, fungus, protozoa, etc. Standard bacterial expression vectors include plasmids such as pBR322 based plasmids, pSKF, pET23D, and commercially available tag-fusion expression systems such as GST and LacZ.

Expression vectors containing regulatory elements from eukaryotic viruses are often used in eukaryotic expression vectors, e.g., SV40 vectors, papilloma virus vectors, and vectors derived from Epstein-Barr virus. Other exemplary eukaryotic vectors include pMSG, pAV009/A+, pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV40 early promoter, SV40 late promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.

The vectors for expressing the guide RNAs can include RNA Pol III promoters to drive expression of the guide RNAs, e.g., the H1, U6 or 7SK promoters. These human promoters allow for expression of gRNAs in mammalian cells following plasmid transfection. Alternatively, a T7 promoter may be used, e.g., for in vitro transcription, and the RNA can be transcribed in vitro and purified. Vectors suitable for the expression of short RNAs, e.g., siRNAs, shRNAs, or other small RNAs, can be used.

Some expression systems have markers for selection of stably transfected cell lines such as thymidine kinase, hygromycin B phosphotransferase, and dihydrofolate reductase. High yield expression systems are also suitable, such as using a baculovirus vector in insect cells, with the gRNA encoding sequence under the direction of the polyhedrin promoter or other strong baculovirus promoters.

The elements that are typically included in expression vectors also include a replicon that functions in E. coli, a gene encoding antibiotic resistance to permit selection of bacteria that harbor recombinant plasmids, and unique restriction sites in nonessential regions of the plasmid to allow insertion of recombinant sequences.

Standard transfection methods are used to produce bacterial, mammalian, yeast or insect cell lines that express large quantities of protein, which are then purified using standard techniques (see, e.g., Colley et al., 1989, J. Biol. Chem., 264:17619-22; Guide to Protein Purification, in Methods in Enzymology, vol. 182 (Deutscher, ed., 1990)). Transformation of eukaryotic and prokaryotic cells are performed according to standard techniques (see, e.g., Morrison, 1977, J. Bacteriol. 132:349-351; Clark-Curtiss & Curtiss, Methods in Enzymology 101:347-362 (Wu et al., eds, 1983).

Any of the known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, nucleofection, liposomes, microinjection, naked DNA, plasmid vectors, viral vectors, both episomal and integrative, and any of the other well-known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell (see, e.g., Sambrook et al., supra). It is only necessary that the particular genetic engineering procedure used be capable of successfully introducing at least one gene into the host cell capable of expressing the gRNA.

The present invention includes the vectors and cells comprising the vectors.

EXAMPLES

The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

Example 1 Assessing Specificity of RNA-Guided Endonucleases

CRISPR RNA-guided nucleases (RGNs) have rapidly emerged as a facile and efficient platform for genome editing. This example describes the use of a human cell-based reporter assay to characterize off-target cleavage of Cas9-based RGNs.

Materials and Methods

The following materials and methods were used in Example 1.

Construction of Guide RNAs

DNA oligonucleotides (Table A) harboring variable 20 nt sequences for Cas9 targeting were annealed to generate short double-strand DNA fragments with 4 bp overhangs compatible with ligation into BsmBI-digested plasmid pMLM3636. Cloning of these annealed oligonucleotides generates plasmids encoding a chimeric +103 single-chain guide RNA with 20 variable 5′ nucleotides under expression of a U6 promoter (Hwang et al., Nat Biotechnol 31, 227-229 (2013); Mali et al., Science 339, 823-826 (2013)). pMLM3636 and the expression plasmid pJDS246 (encoding a codon optimized version of Cas9) used in this study are both available through the non-profit plasmid distribution service Addgene (addgene.org/crispr-cas).

TABLE A EGFP Target Site 1 gRNA Target Sequence Position 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 G G G C A C G G G C A G C T T G C C G G G G G C A C G G G C A G C T T G C C G

G G G C A C G G G C A G C T T G C C

G G G G C A C G G G C A G C T T G C

G G G G G C A C G G G C A G C T T G

C G G G G G C A C G G G C A G C T T

C C G G G G G C A C G G G C A G C T

G C C G G G G G C A C G G G C A G C

T G C C G G G G G C A C G G G C A G

T T G C C G G G G G C A C G G G C A

C T T G C C G G G G G C A C G G G C

G C T T G C C G G G G G C A C G G G

A G C T T G C C G G G G G C A C G G

C A G C T T G C C G G G G G C A C G

G C A G C T T G C C G G G G G C A C

G G C A G C T T G C C G G G G G C A

G G G C A G C T T G C C G G G G G C

C G G G C A G C T T G C C G G G G G

A C G G G C A G C T T G C C G G G G

C A C G G G C A G C T T G C C G G G

G C A C G G G C A G C T T G C C G G G G G C A C G G G C A G C T T G C C

G G G C A C G G G C A G C T T G

G G G G G C A C G G G C A G C T

C C G G G G G C A C G G G C A G

T G C C G G G G G C A C G G G C

C T T G C C G G G G G C A C G G

A G C T T G C C G G G G G C A C

G C A G C T T G C C G G G G G C

G G G C A G C T T G C C G G G G

A C G G G C A G C T T G C C G G G

C A C G G G C A G C T T G C C G G G

A C G G G C A G C T T G C C G G G

C G G G C A G C T T G C C G G G

G G G C A G C T T G C C G G G

G G C A G C T T G C C G G G

G C A G C T T G C C G G G

C A G C T T G C C G G G

A G C T T G C C G G G G G C A C G G G C A G C T T G C

G

G G G C A C G G G C A G  C T T

C

G G G G G C A C G G G C A G C

T G C

G G G G G C A C G G G C A

C T T G C

G G G G G C A C G G G

A G C T T G C

G G G G G C A C G

G C A G C T T G C

G G G G G C A

G G G C A G C T T G C

G G G G G

A C G G G C A G C T T G C

G G G

G C A C G G G C A G C T T G C

G G G G G C A C G G G

A G C T T G C C G

G G G C A C G G G

A G C T T

C C G G G G G C A C G G G

A G C

T G C C G G G G G C A C G G G

A

C T T G C C G G G G G C A C G

G

A G C T T G C C G G G G G C A

G G G

A G C T T G C C G G G G G

A C G G G

A G C T T G C C G G G

G C A C G G G

A G C T T G C C G G G

G C A C G G G C A G C T T G C C G

G

G C A C G G G C A G C T T

C C G G G

G C A C G G G C A G C

T G C C G G G

G C A C G G G C A

C T T G C C G G G

G C A C G

G C A G C T T G C C G G G

G C A

G G G C A G C T T G C C G G G

G

A C G G G C A G C T T G C C G G Oligos for generating gRNA expression plasmid SEQ ID NO: oligonucleotide 1 (5′ to 3′) # oligonucleotide 2 (5′ to 3′) # 2413 ACACCGGGCACGGGCAGCTTGCCGGG  36. AAAACCCGGCAAGCTGCCCGTGCCCG 230. 2414 ACACCGGGCACGGGCAGCTTGCCGCG  37. AAAACGCGGCAAGCTGCCCGTGCCCG 231. 2415 ACACCGGGCACGGGCAGCTTGCCCGG  38. AAAACCGGGCAAGCTGCCCGTGCCCG 232. 2416 ACACCGGGCACGGGCAGCTTGCGGGG  39. AAAACCCCGCAAGCTGCCCGTGCCCG 233. 2417 ACACCGGGCACGGGCAGCTTGGCGGG  40. AAAACCCGCCAAGCTGCCCGTGCCCG 234. 2418 ACACCGGGCACGGGCAGCTTCCCGGG  41. AAAACCCGGGAAGCTGCCCGTGCCCG 235. 2419 ACACCGGGCACGGGCAGCTAGCCGGG  42. AAAACCCGGCTAGCTGCCCGTGCCCG 236. 2420 ACACCGGGCACGGGCAGCATGCCGGG  43. AAAACCCGGCATGCTGCCCGTGCCCG 237. 2421 ACACCGGGCACGGGCAGGTTGCCGGG  44. AAAACCCGGCAACCTGCCCGTGCCCG 238. 2422 ACACCGGGCACGGGCACCTTGCCGGG  45. AAAACCCGGCAAGGTGCCCGTGCCCG 239. 2423 ACACCGGGCACGGGCTGCTTGCCGGG  46. AAAACCCGGCAAGCAGCCCGTGCCCG 240. 2424 ACACCGGGCACGGGGAGCTTGCCGGG  47. AAAACCCGGCAAGCTCCCCGTGCCCG 241. 2425 ACACCGGGCACGGCCAGCTTGCCGGG  48. AAAACCCGGCAAGCTGGCCGTGCCCG 242. 2426 ACACCGGGCACGCGCAGCTTGCCGGG  49. AAAACCCGGCAAGCTGCGCGTGCCCG 243. 2427 ACACCGGGCACCGGCAGCTTGCCGGG  50. AAAACCCGGCAAGCTGCCGGTGCCCG 244. 2428 ACACCGGGCAGGGGCAGCTTGCCGGG  51. AAAACCCGGCAAGCTGCCCCTGCCCG 245. 2429 ACACCGGGCTCGGGCAGCTTGCCGGG  52. AAAACCCGGCAAGCTGCCCGAGCCCG 246. 2430 ACACCGGGGACGGGCAGCTTGCCGGG  53. AAAACCCGGCAAGCTGCCCGTCCCCG 247. 2431 ACACCGGCCACGGGCAGCTTGCCGGG  54. AAAACCCGGCAAGCTGCCCGTGGCCG 248. 2432 ACACCGCGCACGGGCAGCTTGCCGGG  55. AAAACCCGGCAAGCTGCCCGTGCGCG 249. 2433 ACACCGGGCACGGGCAGCTTGCCCCG  56. AAAACGGGGCAAGCTGCCCGTGCCCG 250. 2434 ACACCGGGCACGGGCAGCTTGGGGGG  57. AAAACCCCCCAAGCTGCCCGTGCCCG 251. 2435 ACACCGGGCACGGGCAGCTACCCGGG  58. AAAACCCGGGTAGCTGCCCGTGCCCG 252. 2436 ACACCGGGCACGGGCAGGATGCCGGG  59. AAAACCCGGCATCCTGCCCGTGCCCG 253. 2437 ACACCGGGCACGGGCTCCTTGCCGGG  60. AAAACCCGGCAAGGAGCCCGTGCCCG 254. 2438 ACACCGGGCACGGCGAGCTTGCCGGG  61. AAAACCCGGCAAGCTCGCCGTGCCCG 255. 2439 ACACCGGGCACCCGCAGCTTGCCGGG  62. AAAACCCGGCAAGCTGCGGGTGCCCG 256. 2440 ACACCGGGCTGGGGCAGCTTGCCGGG  63. AAAACCCGGCAAGCTGCCCCAGCCCG 257. 2441 ACACCGGCGACGGGCAGCTTGCCGGG  64. AAAACCCGGCAAGCTGCCCGTCGCCG 258. 2442 ACACCGCCCACGGGCAGCTTGCCGGG  65. AAAACCCGGCAAGCTGCCCGTGCGGG 259. 2443 ACACCGCCGACGGGCAGCTTGCCGGG  66. AAAACCCGGCAAGCTGCCCGTGCCCG 260. 2444 ACACCGCCGTCGGGCAGCTTGCCGGG  67. AAAACCCGGCAAGCTGCCCGTGCCCG 261. 2445 ACACCGCCGTGGGGCAGCTTGCCGGG  68. AAAACCCGGCAAGCTGCCCGTGCCCG 262. 2446 ACACCGCCGTGCGGCAGCTTGCCGGG  69. AAAACCCGGCAAGCTGCCCGTGCCCG 263. 2447 ACACCGCCGTGCCGCAGCTTGCCGGG  70. AAAACCCGGCAAGCTGCCCGTGCCCG 264. 2448 ACACCGCCGTGCCCCAGCTTGCCGGG  71. AAAACCCGGCAAGCTGCCCGTGCCCG 265. 2449 ACACCGCCGTGCCCGAGCTTGCCGGG  72. AAAACCCGGCAAGCTGCCCGTGCCCG 266. 2450 ACACCGGGCACGGGCAGCTTGCGGCG  73. AAAACGCCGCAAGCTGCCCGTGCCCG 267. 2451 ACACCGGGCACGGGCAGCTTCCGGGG  74. AAAACCCCGGAAGCTGCCCGTGCCCG 268. 2452 ACACCGGGCACGGGCAGCATGCGGGG  75. AAAACCCCGCATGCTGCCCGTGCCCG 269. 2453 ACACCGGGCACGGGCACCTTGCGGGG  76. AAAACCCCGCAAGGTGCCCGTGCCCG 270. 2454 ACACCGGGCACGGGGAGCTTGCGGGG  77. AAAACCCCGCAAGCTCCCCGTGCCCG 271. 2455 ACACCGGGCACGCGCAGCTTGCGGGG  78. AAAACCCCGCAAGCTGCGCGTGCCCG 272. 2456 ACACCGGGCAGGGGCAGCTTGCGGGG  79. AAAACCCCGCAAGCTGCCCCTGCCCG 273. 2457 ACACCGGGGACGGGCAGCTTGCGGGG  80. AAAACCCCGCAAGCTGCCCGTCCCCG 274. 2458 ACACCGCGCACGGGCAGCTTGCGGGG  81. AAAACCCCGCAAGCTGCCCGTGCGCG 275. 2459 ACACCGGGCACGGGGAGCATGCCGGG  82. AAAACGCGGCAAGCTCCCCGTGCCCG 276. 2460 ACACCGGGCACGGGGAGCTTCCCGGG  83. AAAACCCGGGAAGCTCCCCGTGCCCG 277. 2461 ACACCGGGCACGGGGAGCATGCCGGG  84. AAAACCCGGCATGCTCCCCGTGCCCG 278. 2462 ACACCGGGCACGGGGACCTTGCCGGG  85. AAAACCCGGCAAGGTCCCCGTGCCCG 279. 2463 ACACCGGGCACGCGGAGCTTGCCGGG  86. AAAACCCGGCAAGCTCCGCGTGCCCG 280. 2464 ACACCGGGCAGGGGGAGCTTGCCGGG  87. AAAACCCGGCAAGCTCCCCCTGCCCG 281. 2465 ACACCGGGGACGGGGAGCTTGCCGGG  88. AAAACCCGGCAAGCTCCCCGTCCCCG 282. 2466 ACACCGCGCACGGGGAGCTTGCCGGG  89. AAAACCCGGCAAGCTCCCCGTGCGCG 283. 2467 ACACCGCGCACGGGGAGCTTGCCGGG  90. AAAACGCGGCAAGCTGCCCGTGCGCG 284. 2468 ACACCGCGCACGGGCAGCTTCCCGGG  91. AAAACCCGGGAAGCTGCCCGTGCGCG 285. 2469 ACACCGCGCACGGGCAGCATGCCGGG  92. AAAACCCGGCATGCTGCCCGTGCGCG 286. 2470 ACACCGCGCACGGGCACCTTGCCGGG  93. AAAACCCGGCAAGGTGCCCGTGCGCG 287. 2471 ACACCGCGCACGCGCAGCTTGCCGGG  94. AAAACCCGGCAAGCTGCGCGTGCGCG 288. 2472 ACACCGCGCAGGGGCAGCTTGCCGGG  95. AAAACCCGGCAAGCTGCCCCTGCGCG 289. 2473 ACACCGCGGACGGGCAGCTTGCCGGG  96. AAAACCCGGCAAGCTGCCCGTCCGCG 290. EGFP Target Site 2 gRNA Target Sequence Position 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 G A T G C C G T T C T T C T G C T T G T G A T G C C G T T C T T C T G C T T G

G A T G C C G T T C T T C T G C T T

T G A T G C C G T T C T T C T G C T

G T G A T G C C G T T C T T C T G C

T G T G A T G C C G T T C T T C T G

T T G T G A T G C C G T T C T T C T

C T T G T G A T G C C G T T C T T C

G C T T G T G A T G C C G T T C T T

T G C T T G T G A T G C C G T T C T

C T G C T T G T G A T G C C G T T C

T C T G C T T G T G A T G C C G T T

T T C T G C T T G T G A T G C C G T

C T T C T G C T T G T G A T G C C G

T C T T C T G C T T G T G A T G C C

T T C T T C T G C T T G T G A T G C

G T T C T T C T G C T T G T G A T G

C G T T C T T C T G C T T G T G A T

C C G T T C T T C T G C T T G T G A

G C C G T T C T T C T G C T T G T G

T G C C G T T C T T C T G C T T G T G A T G C C G T T C T T C T G C T T

G A T G C C G T T C T T C T G C

G T G A T G C C G T T C T T C T

T T G T G A T G C C G T T C T T

G C T T G T G A T G C C G T T C

C T G C T T G T G A T G C C G T

T T C T G C T T G T G A T G C C

T C T T C T G C T T G T G A T G

G T T C T T C T G C T T G T G A

C C G T T C T T C T G C T T G T G

G C C G T T C T T C T G C T T G T G

C C G T T C T T C T G C T T G T G

C G T T C T T C T G C T T G T G

G T T C T T C T G C T T G T G

T T C T T C T G C T T G T G

T C T T C T G C T T G T G

C T T C T G C T T G T G

T T C T G C T T G T G A T G C C G T T C T T C T G C T

G

G A T G C C G T T C T T C T G

T

G T G A T G C C G T T C T T C

G C T

G T G A T G C C G T T C T

C T G C T

G T G A T G C C G T T

T T C T G C T

G T G A T G C C G

T C T T C T G C T

G T G A T G C

G T T C T T C T G C T

G T G A T

C C G T T C T T C T G C T

G T G

T G C C G T T C T T C T G C T

G T G A T G C C G T T

T T C T G C T T G

G A T G C C G T T

T T C T G

T T G T G A T G C C G T T

T T C

G C T T G T G A T G C C G T T

T

C T G C T T G T G A T G C C G

T

T T C T G C T T G T G A T G C

G T T

T T C T G C T T G T G A T

C C G T T

T T C T G C T T G T G

T G C C G T T

T T C T G C T T G T G

T G C C G T T C T T C T G C T T G

G

T G C C G T T C T T C T G

T T G T G

T G C C G T T C T T C

G C T T G T G

T G C C G T T C T

C T G C T T G T G

T G C C G

T C T T C T G C T T G T G

T G C

G T T C T T C T G C T T G T G

T

C C G T T C T T C T G C T T G T Oligos for generating gRNA expression plasmid SEQ ID NO: oligonucleotide 1 (5′ to 3′) oligonucleotide 2 (5′ to 3′) 2474 ACACCGATGCCGTTCTTCTGCTTGTG  97. AAAACACAAGCAGAAGAACGGCATCG 291. 2475 ACACCGATGCCGTTCTTCTGCTTGAG  98. AAAACACAAGCAGAAGAACGGCATCG 292. 2476 ACACCGATGCCGTTCTTCTGCTTCTG  99. AAAACACAAGCAGAAGAACGGCATCG 293. 2477 ACACCGATGCCGTTCTTCTGCTAGTG 100. AAAACACAAGCAGAAGAACGGCATCG 294. 2478 ACACCGATGCCGTTCTTCTGCATGTG 101. AAAACACAAGCAGAAGAACGGCATCG 295. 2479 ACACCGATGCCGTTCTTCTGGTTGTG 102. AAAACACAAGCAGAAGAACGGCATCG 296. 2480 ACACCGATGCCGTTCTTCTCCTTGTG 103. AAAACACAAGCAGAAGAACGGCATCG 297. 2481 ACACCGATGCCGTTCTTCAGCTTGTG 104. AAAACACAAGCAGAAGAACGGCATCG 298. 2482 ACACCGATGCCGTTCTTGTGCTTGTG 105. AAAACACAAGCAGAAGAACGGCATCG 299. 2483 ACACCGATGCCGTTCTACTGCTTGTG 106. AAAACACAAGCAGAAGAACGGCATCG 300. 2484 ACACCGATGCCGTTCATCTGCTTGTG 107. AAAACACAAGCAGAAGAACGGCATCG 301. 2485 ACACCGATGCCGTTGTTCTGCTTGTG 108. AAAACACAAGCAGAAGAACGGCATCG 302. 2486 ACACCGATGCCGTACTTCTGCTTGTG 109. AAAACACAAGCAGAAGAACGGCATCG 303. 2487 ACACCGATGCCGATCTTCTGCTTGTG 110. AAAACACAAGCAGAAGAACGGCATCG 304. 2488 ACACCGATGCCCTTCTTCTGCTTGTG 111. AAAACACAAGCAGAAGAACGGCATCG 305. 2489 ACACCGATGCGGTTCTTCTGCTTGTG 112. AAAACACAAGCAGAAGAACGGCATCG 306. 2490 ACACCGATGGCGTTCTTCTGCTTGTG 113. AAAACACAAGCAGAAGAACGGCATCG 307. 2491 ACACCGATCCCGTTCTTCTGCTTGTG 114. AAAACACAAGCAGAAGAACGGCATCG 308. 2492 ACACCGAAGCCGTTCTTCTGCTTGTG 115. AAAACACAAGCAGAAGAACGGCATCG 309. 2493 ACACCGTTGCCGTTCTTCTGCTTGTG 116. AAAACACAAGCAGAAGAACGGCATCG 310. 2494 ACACCGATGCCGTTCTTCTGCTTCAG 117. AAAACTGAAGCAGAAGAACGGCATCG 311. 2495 ACACCGATGCCGTTCTTCTGCAAGTG 118. AAAACACAAGCAGAAGAACGGCATCG 312. 2496 ACACCGATGCCGTTCTTCTCGTTGTG 119. AAAACACAAGCAGAAGAACGGCATCG 313. 2497 ACACCGATGCCGTTCTTGAGCTTGTG 120. AAAACACAAGCTCAAGAACGGCATCG 314. 2498 ACACCGATGCCGTTCAACTGCTTGTG 121. AAAACACAAGCAGAAGAACGGCATCG 315. 2499 ACACCGATGCCGTAGTTCTGCTTGTG 122. AAAACACAAGCAGAAGAACGGCATCG 316. 2500 ACACCGATGCCCATCTTCTGCTTGTG 123. AAAACACAAGCAGAAGAACGGCATCG 317. 2501 ACACCGATGGGGTTCTTCTGCTTGTG 124. AAAACACAAGCAGAAGAACGGCATCG 318. 2502 ACACCGAACCCGTTCTTCTGCTTGTG 125. AAAACACAAGCAGAAGAACGGCATCG 319. 2503 ACACCGTAGCCGTTCTTCTGCTTGTG 126. AAAACACAAGCAGAAGAACGGCAAGG 320. 2504 ACACCGTACCCGTTCTTCTGCTTGTG 127. AAAACACAAGCAGAAGAACGGGTACG 321. 2505 ACACCGTACGCGTTCTTCTGCTTGTG 128. AAAACACAAGCAGAAGAACGCGTACG 322. 2506 ACACCGTACGGGTTCTTCTGCTTGTG 129. AAAACACAAGCAGAAGAACCCGTACG 323. 2507 ACACCGTACGGCTTCTTCTGCTTGTG 130. AAAACACAAGCAGAAGAAGCCGTACG 324. 2508 ACACCGTACGGCATCTTCTGCTTGTG 131. AAAACACAAGCAGAAGATGCCGTACG 325. 2509 ACACCGTACGGCAACTTCTGCTTGTG 132. AAAACACAAGCAGAAGTTGCCGTACG 326. 2510 ACACCGTACGGCAAGTTCTGCTTGTG 133. AAAACACAAGCAGAACTTGCCGTACG 327. 2511 ACACCGATGCCGTTCTTCTGCTAGAG 134. AAAACTCTAGCAGAAGAACGGCATCG 328. 2512 ACACCGATGCCGTTCTTCTGGTAGTG 135. AAAACACTACCAGAAGAACGGCATCG 329. 2513 ACACCGATGCCGTTCTTCAGCTAGTG 136. AAAACACTAGCTGAAGAACGGCATCG 330. 2514 ACACCGATGCCGTTCTACTGCTAGTG 137. AAAACACTAGCAGTAGAACGGCATCG 331. 2515 ACACCGATGCCGTTGTTCTGCTAGTG 138. AAAACACTAGCAGAACAACGGCATCG 332. 2516 ACACCGATGCCGATCTTCTGCTAGTG 139. AAAACACTAGCAGAAGATCGGCATCG 333. 2517 ACACCGATGCGGTTCTTCTGCTAGTG 140. AAAACACTAGCAGAAGAACCGCATCG 334. 2518 ACACCGATCCCGTTCTTCTGCTAGTG 141. AAAACACTAGCAGAAGAACGGGATCG 335. 2519 ACACCGTTGCCGTTCTTCTGCTAGTG 142. AAAACACTAGCAGAAGAACGGCAACG 336. 2520 ACACCGATGCCGTTGTTCTGCTTGAG 143. AAAACTCAAGCAGAACAACGGCATCG 337. 2521 ACACCGATGCCGTTGTTCTGGTTGTG 144. AAAACACAACCAGAACAACGGCATCG 338. 2522 ACACCGATGCCGTTGTTCAGCTTGTG 145. AAAACACAAGCTGAACAACGGCATCG 339. 2523 ACACCGATGCCGTTGTACTGCTTGTG 146. AAAACACAAGCAGTACAACGGCATCG 340. 2524 ACACCGATGCCGATGTTCTGCTTGTG 147. AAAACACAAGCAGAACATCGGCATCG 341. 2525 ACACCGATGCGGTTGTTCTGCTTGTG 148. AAAACACAAGCAGAACAACCGCATCG 342. 2526 ACACCGATCCCGTTGTTCTGCTTGTG 149. AAAACACAAGCAGAACAACGGGATCG 343. 2527 ACACCGTTGCCGTTGTTCTGCTTGTG 150. AAAACACAAGCAGAACAACGGCAACG 344. 2528 ACACCGTTGCCGTTCTTCTGCTTGAG 151. AAAACTCAAGCAGAAGAACGGCAACG 345. 2529 ACACCGTTGCCGTTCTTCTGGTTGTG 152. AAAACACAACCAGAAGAACGGCAACG 346. 2530 ACACCGTTGCCGTTCTTCAGCTTGTG 153. AAAACACAAGCTGAAGAACGGCAACG 347. 2531 ACACCGTTGCCGTTCTACTGCTTGTG 154. AAAACACAAGCAGTAGAACGGCAACG 348. 2532 ACACCGTTGCCGATCTTCTGCTTGTG 155. AAAACACAAGCAGAAGATCGGCAACG 349. 2533 ACACCGTTGCGGTTCTTCTGCTTGTG 156. AAAACACAAGCAGAAGAACCGCAACG 350. 2534 ACACCGTTCCCGTTCTTCTGCTTGTG 157. AAAACACAAGCAGAAGAACGGGAACG 351. EGFP Target Site 3 gRNA Target Sequence Position 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 G G T G G T G C A G A T G A A C T T C A G G T G G T G C A G A T G A A C T T C

G G T G G T G C A G A T G A A C T T

A G G T G G T G C A G A T G A A C T

C A G G T G G T G C A G A T G A A C

T C A G G T G G T G C A G A T G A A

T T C A G G T G G T G C A G A T G A

C T T C A G G T G G T G C A G A T G

A C T T C A G G T G G T G C A G A T

A A C T T C A G G T G G T G C A G A

G A A C T T C A G G T G G T G C A G

T G A A C T T C A G G T G G T G C A

A T G A A C T T C A G G T G G T G C

G A T G A A C T T C A G G T G G T G

A G A T G A A C T T C A G G T G G T

C A G A T G A A C T T C A G G T G G

G C A G A T G A A C T T C A G G T G

T G C A G A T G A A C T T C A G G T

G T G C A G A T G A A C T T C A G G

G G T G C A G A T G A A C T T C A G

T G G T G C A G A T G A A C T T C A G G T G G T G C A G A T G A A C T T

G G T G G T G C A G A T G A A C

C A G G T G G T G C A G A T G A

T T C A G G T G G T G C A G A T

A C T T C A G G T G G T G C A G

G A A C T T C A G G T G G T G C

A T G A A C T T C A G G T G G T

A G A T G A A C T T C A G G T G

G C A G A T G A A C T T C A G G

G T G C A G A T G A A C T T C A G

G G T G C A G A T G A A C T T C A G

G T G C A G A T G A A C T T C A G

T G C A G A T G A A C T T C A G

G C A G A T G A A C T T C A G

C A G A T G A A C T T C A G

A G A T G A A C T T C A G

G A T G A A C T T C A G

A T G A A C T T C A G G T G G T G C A G A T G A A C T

C

G G T G G T G C A G A T G A A

T

C A G G T G G T G C A G A T G

A C T

C A G G T G G T G C A G A

G A A C T

C A G G T G G T G C A

A T G A A C T

C A G G T G G T G

A G A T G A A C T

C A G G T G G

G C A G A T G A A C T

C A G G T

G T G C A G A T G A A C T

C A G

T G G T G C A G A T G A A C T

C A G G T G G T G C A

A T G A A C T T C

G G T G G T G C A

A T G A A

T T C A G G T G G T G C A

A T G

A C T T C A G G T G G T G C A

A

G A A C T T C A G G T G G T G

A

A T G A A C T T C A G G T G G

G C A

A T G A A C T T C A G G T

G T G C A

A T G A A C T T C A G

T G G T G C A

A T G A A C T T C A G

T G G T G C A G  A T G A A C T T C

G

T G G T G C A G A T G A A

T T C A G

T G G T G C A G A T G

A C T T C A G

T G G T G C A G A

G A A C T T C A G

T G G T G

A G A T G A A C T T C A G

T G G

G C A G A T G A A C T T C A G

T

G T G C A G A T G A A C T T C A Oligos for generating gRNA expression plasmid SEQ ID NO: oligonucleotide 1 (5′ to 3′) oligonucleotide 2 (5′ to 3′) 2535 ACACCGGTGGTGCAGATGAACTTCAG 158. AAAACTGAAGTTCATCTGCACCACCG 352. 2536 ACACCGGTGGTGCAGATGAACTTCTG 159. AAAACAGAAGTTCATCTGCACCACCG 353. 2537 ACACCGGTGGTGCAGATGAACTTGAG 160. AAAACTCAAGTTCATCTGCACCACCG 354. 2538 ACACCGGTGGTGCAGATGAACTACAG 161. AAAACTGTAGTTCATCTGCACCACCG 355. 2539 ACACCGGTGGTGCAGATGAACATCAG 162. AAAACTGATGTTCATCTGCACCACCG 356. 2540 ACACCGGTGGTGCAGATGAAGTTCAG 163. AAAACTGAACTTCATCTGCACCACCG 357. 2541 ACACCGGTGGTGCAGATGATCTTCAG 164. AAAACTGAAGATCATCTGCACCACCG 358. 2542 ACACCGGTGGTGCAGATGTACTTCAG 165. AAAACTGAAGTACATCTGCACCACCG 359. 2543 ACACCGGTGGTGCAGATCAACTTCAG 166. AAAACTGAAGTTGATCTGCACCACCG 360. 2544 ACACCGGTGGTGCAGAAGAACTTCAG 167. AAAACTGAAGTTCTTCTGCACCACCG 361. 2545 ACACCGGTGGTGCAGTTGAACTTCAG 168. AAAACTGAAGTTCAACTGCACCACCG 362. 2546 ACACCGGTGGTGCACATGAACTTCAG 169. AAAACTGAAGTTCATGTGCACCACCG 363. 2547 ACACCGGTGGTGCTGATGAACTTCAG 170. AAAACTGAACTTCATCTGCACCACCG 364. 2548 ACACCGGTGGTGGAGATGAACTTCAG 171. AAAACTGAAGTTCATCTCCACCACCG 365. 2549 ACACCGGTGGTCCAGATGAACTTCAG 172. AAAACTGAAGTTCATCTGGACCACCG 366. 2550 ACACCGGTGGAGCAGATGAACTTCAG 173. AAAACTGAAGTTCATCTGCTCCACCG 367. 2551 ACACCGGTGCTGCAGATGAACTTCAG 174. AAAACTGAAGTTCATCTGCAGCACCG 368. 2552 ACACCGGTCGTGCAGATGAACTTCAG 175. AAAACTGAAGTTCATCTGCACGACCG 369. 2553 ACACCGGAGGTGCAGATGAACTTCAG 176. AAAACTGAAGTTCATCTGCACCTCCG 370. 2554 ACACCGCTGGTGCAGATGAACTTCAG 177. AAAACTGAAGTTCATCTGCACCAGCG 371. 2555 ACACCGGTGGTGCAGATGAACTTGTG 178. AAAACACAAGTTCATCTGCACCACCG 372. 2556 ACACCGGTGGTGCAGATGAACAACAG 179. AAAACTGTTGTTCATCTGCACCACCG 373. 2557 ACACCGGTGGTGCAGATGATGTTCAG 180. AAAACTGAACATCATCTGCACCACCG 374. 2558 ACACCGGTGGTGCAGATCTACTTCAG 181. AAAACTGAAGTAGATCTGCACCACCG 375. 2559 ACACCGGTGGTGCAGTAGAACTTCAG 182. AAAACTGAAGTTCTACTGCACCACCG 376. 2560 ACACCGGTGGTGCTCATGAACTTCAG 183. AAAACTGAAGTTCATGAGCACCACCG 377. 2561 ACACCGGTGGTCGAGATGAACTTCAG 184. AAAACTGAAGTTCATCTCGACCACCG 378. 2562 ACACCGGTGCAGCAGATGAACTTCAG 185. AAAACTGAAGTTCATCTGCTGCACCG 379. 2563 ACACCGGACGTGCAGATGAACTTCAG 186. AAAACTGAAGTTCATCTGCACGTCCG 380. 2564 ACACCGCAGGTGCAGATGAACTTCAG 187. AAAACTGAAGTTCATCTGCACCAGGG 381. 2565 ACACCGCACGTGCAGATGAACTTCAG 188. AAAACTGAAGTTCATCTGCACGTGCG 382. 2566 ACACCGCACCTGCAGATGAACTTCAG 189. AAAACTGAAGTTCATCTGCAGGTGCG 383. 2567 ACACCGCACCAGCAGATGAACTTCAG 190. AAAACTGAAGTTCATCTGCTGGTGCG 384. 2568 ACACCGCACCACCAGATGAACTTCAG 191. AAAACTGAAGTTCATCTGGTGGTGCG 385. 2569 ACACCGCACCACGAGATGAACTTCAG 192. AAAACTGAAGTTCATCTCGTGGTGCG 386. 2570 ACACCGCACCACGTGATGAACTTCAG 193. AAAACTGAAGTTCATCACGTGGTGCG 387. 2571 ACACCGCACCACGTCATGAACTTCAG 194. AAAACTGAAGTTCATGACGTGGTGCG 388. 2572 ACACCGGTGGTGCAGATGAACTACTG 195. AAAACAGTAGTTCATCTGCACCACCG 389. 2573 ACACCGGTGGTGCAGATGAAGTACAG 196. AAAACTGTACTTCATCTGCACCACCG 390. 2574 ACACCGGTGGTGCAGATGTACTACAG 197. AAAACTGTAGTACATCTGCACCACCG 391. 2575 ACACCGGTGGTGCAGAAGAACTACAG 198. AAAACTGTAGTTCTTCTGCACCACCG 392. 2576 ACACCGGTGGTGCACATGAACTACAG 199. AAAACTGTAGTTCATGTGCACCACCG 393. 2577 ACACCGGTGGTGGAGATGAACTACAG 200. AAAACTGTAGTTCATCTCCACCACCG 394. 2578 ACACCGGTGGAGCAGATGAACTACAG 201. AAAACTGTAGTTCATCTGCTCCACCG 395. 2579 ACACCGGTCGTGCAGATGAACTACAG 202. AAAACTGTAGTTCATCTGCACGACCG 396. 2580 ACACCGCTGGTGCAGATGAACTACAG 203. AAAACTGTAGTTCATCTGCACCAGCG 397. 2581 ACACCGGTGGTGCACATGAACTTCTG 204. AAAACAGAAGTTCATGTGCACCACCG 398. 2582 ACACCGGTGGTGCACATGAAGTTCAG 205. AAAACTGAACTTCATGTGCACCACCG 399. 2583 ACACCGGTGGTGCACATGTACTTCAG 206. AAAACTGAAGTACATGTGCACCACCG 400. 2584 ACACCGGTGGTGCACAAGAACTTCAG 207. AAAACTGAAGTTCTTGTGCACCACCG 401. 2585 ACACCGGTGGTGGACATGAACTTCAG 208. AAAACTGAAGTTCATGTCCACCACCG 402. 2586 ACACCGGTGGAGCACATGAACTTCAG 209. AAAACTGAAGTTCATGTGCTCCACCG 403. 2587 ACACCGGTCGTGCACATGAACTTCAG 210. AAAACTGAAGTTCATGTGCACGACCG 404. 2588 ACACCGCTGGTGCACATGAACTTCAG 211. AAAACTGAAGTTCATGTGCACCAGCG 405. 2589 ACACCGCTGGTGCAGATGAACTTCTG 212. AAAACAGAAGTTCATCTGCACCAGCG 406. 2590 ACACCGCTGGTGCAGATGAAGTTCAG 213. AAAACTGAACTTCATCTGCACCAGCG 407. 2591 ACACCGCTGGTGCAGATGTACTTCAG 214. AAAACTGAAGTACATCTGCACCAGCG 408. 2592 ACACCGCTGGTGCAGAAGAACTTCAG 215. AAAACTGAAGTTCTTCTGCACCAGCG 409. 2593 ACACCGCTGGTGGAGATGAACTTCAG 216. AAAACTGAAGTTCATCTCCACCAGCG 410. 2594 ACACCGCTGGAGCAGATGAACTTCAG 217. AAAACTGAAGTTCATCTGCTCCAGCG 411. 2595 ACACCGCTCGTGCAGATGAACTTCAG 218. AAAACTGAAGTTCATCTGCACGAGCG 412. Endogenous Target 1 (VEGFA Site 1) 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 G G G T G G G G G G A G T T T G C T C C Oligos for generating gRNA expression plasmid SEQ ID NO: oligonucleotide 1 (5′ to 3′) # oligonucleotide 2 (5′ to 3′) # 2596 ACACCGGGTGGGGGGAGTTTGCTCCG 219. AAAACGGAGCAAACTCCCCCCACCCG 413. 220. 414. Endogenous Target 2 (VEGFA Site 2): 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 G A C C C C C T C C A C C C C G C C T C Oligos for generating gRNA expression plasmid SEQ ID NO: oligonucleotide 1 (5′ to 3′) oligonucleotide 2 (5′ to 3′) 2597 ACACCGACCCCCTCCACCCCGCCTCG 221. AAAACGAGGCGGGGTGGAGGGGGTCG 415. 222. 416. Endogenous Target 3 (VEGFA Site 3): 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 G G T G A G T G A G T G T G T G C G T G Oligos for generating gRNA expression plasmid SEQ ID NO: oligonucleotide 1 (5′ to 3′) oligonucleotide 2 (5′ to 3′) 2598 ACACCGGTGAGTGAGTGTGTGCGTGG 223. AAAACCACGCACACACTCACTCACCG 417. 224. 418. Endogenous Target 4 (EMX1): 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 G A G T C C G A G C A G A A G A A G A A Oligos for generating gRNA expression plasmid SEQ ID NO: oligonucleotide 1 (5′ to 3′) oligonucleotide 2 (5′ to 3′) 2599 ACACCGAGTCCGAGCAGAAGAAGAAG 225. AAAACTTCTTCTTCTGCTCGGACTCG 419. 226. 420. Endogenous Target 5 (RNF2): 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 G T C A T C T T A G T C A T T A C C T G Oligos for generating gRNA expression plasmid SEQ ID NO: oligonucleotide 1 (5′ to 3′) oligonucleotide 2 (5′ to 3′) 2600 ACACCGTCATCTTAGTCATTACCTGG 227. AAAACCAGGTAATGACTAAGATGACG 421. 228. 422. Endogenous Target 6 (FANCF): 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 G G A A T C C C T T C T G C A G C A C C Oligos for generating gRNA expression plasmid SEQ ID NO: oligonucleotide 1 (5′ to 3′) oligonucleotide 2 (5′ to 3′) 2601 ACACCGGAATCCCTTCTGCAGCACCG 229. AAAACGGTGCTGCAGAAGGGATTCCG 423. Sequences of oligonucleotides used to generate expression plasmids encoding single gRNAs/variant single gRNAs targeted to sites in the EGFP reporter gene and single gRNAs targeted to six endogenous human gene targets. #, SEQ ID NO:.

EGFP Activity Assays

U2OS.EGFP cells harboring a single integrated copy of an EGFP-PEST fusion gene were cultured as previously described (Reyon et al., Nat Biotech 30, 460-465 (2012)). For transfections, 200,000 cells were Nucleofected with the indicated amounts of sgRNA expression plasmid and pJDS246 together with 30 ng of a Td-tomato-encoding plasmid using the SE Cell Line 4D-Nucleofector™ X Kit (Lonza) according to the manufacturer's protocol. Cells were analyzed 2 days post-transfection using a BD LSRII flow cytometer. Transfections for optimizing gRNA/Cas9 plasmid concentration were performed in triplicate and all other transfections were performed in duplicate.

PCR Amplification and Sequence Verification of Endogenous Human Genomic Sites

PCR reactions were performed using Phusion Hot Start II high-fidelity DNA polymerase (NEB) with PCR primers and conditions listed in Table B. Most loci amplified successfully using touchdown PCR (98° C., 10 s; 72-62° C., −1° C./cycle, 15 s; 72° C., 30 s]10 cycles, [98° C., 10 s; 62° C., 15 s; 72° C., 30 s]25 cycles). PCR for the remaining targets were performed with 35 cycles at a constant annealing temperature of 68° C. or 72° C. and 3% DMSO or 1M betaine, if necessary. PCR products were analyzed on a QIAXCEL capillary electrophoresis system to verify both size and purity. Validated products were treated with ExoSap-IT (Affymetrix) and sequenced by the Sanger method (MGH DNA Sequencing Core) to verify each target site.

TABLE B

Sequences and characteristics of genomic on- and off-target sites for six RGNs targeted to endogenous human genes and primers and PCR conditions used to amplify these sites.

Determination of RGN-Induced on- and Off-Target Mutation Frequencies in Human Cells

For U2OS.EGFP and K562 cells, 2×10⁵ cells were transfected with 250 ng of gRNA expression plasmid or an empty U6 promoter plasmid (for negative controls), 750 ng of Cas9 expression plasmid, and 30 ng of td-Tomato expression plasmid using the 4D Nucleofector System according to the manufacturer's instructions (Lonza). For HEK293 cells, 1.65×10⁵ cells were transfected with 125 ng of gRNA expression plasmid or an empty U6 promoter plasmid (for the negative control), 375 ng of Cas9 expression plasmid, and 30 ng of a td-Tomato expression plasmid using Lipofectamine LTX reagent according to the manufacturer's instructions (Life Technologies). Genomic DNA was harvested from transfected U2OS.EGFP, HEK293, or K562 cells using the QIAamp DNA Blood Mini Kit (QIAGEN), according to the manufacturer's instructions. To generate enough genomic DNA to amplify the off-target candidate sites, DNA from three Nucleofections (for U2OS.EGFP cells), two Nucleofections (for K562 cells), or two Lipofectamine LTX transfections was pooled together before performing T7EI. This was done twice for each condition tested, thereby generating duplicate pools of genomic DNA representing a total of four or six individual transfections. PCR was then performed using these genomic DNAs as templates as described above and purified using Ampure XP beads (Agencourt) according to the manufacturer's instructions. T7EI assays were performed as previously described (Reyon et al., 2012, supra).

DNA Sequencing of NHEJ-Mediated Indel Mutations

Purified PCR products used for the T7EI assay were cloned into Zero Blunt TOPO vector (Life Technologies) and plasmid DNAs were isolated using an alkaline lysis miniprep method by the MGH DNA Automation Core. Plasmids were sequenced using an M13 forward primer (5′-GTAAAACGACGGCCAG-3′ (SEQ ID NO:1059) by the Sanger method (MGH DNA Sequencing Core).

Example 1a Single Nucleotide Mismatches

To begin to define the specificity determinants of RGNs in human cells, a large-scale test was performed to assess the effects of systematically mismatching various positions within multiple gRNA/target DNA interfaces. To do this, a quantitative human cell-based enhanced green fluorescent protein (EGFP) disruption assay previously described (see Methods above and Reyon et al., 2012, supra) that enables rapid quantitation of targeted nuclease activities (FIG. 2B) was used. In this assay, the activities of nucleases targeted to a single integrated EGFP reporter gene can be quantified by assessing loss of fluorescence signal in human U2OS.EGFP cells caused by inactivating frameshift insertion/deletion (indel) mutations introduced by error prone non-homologous end-joining (NHEJ) repair of nuclease-induced double-stranded breaks (DSBs) (FIG. 2B). For the studies described here, three ˜100 nt single gRNAs targeted to different sequences within EGFP were used, as follows:

(SEQ ID NO: 9) EGFP Site 1 GGGCACGGGCAGCTTGCCGGTGG (SEQ ID NO: 10) EGFP Site 2 GATGCCGTTCTTCTGCTTGTCGG (SEQ ID NO: 11) EGFP Site 3 GGTGGTGCAGATGAACTTCAGGG Each of these gRNAs can efficiently direct Cas9-mediated disruption of EGFP expression (see Example 1e and 2a, and FIGS. 3E (top) and 3F (top)).

In initial experiments, the effects of single nucleotide mismatches at 19 of 20 nucleotides in the complementary targeting region of three EGFP-targeted gRNAs were tested. To do this, variant gRNAs were generated for each of the three target sites harboring Watson-Crick transversion mismatches at positions 1 through 19 (numbered 1 to 20 in the 3′ to 5′ direction; see FIG. 1) and the abilities of these various gRNAs to direct Cas9-mediated EGFP disruption in human cells tested (variant gRNAs bearing a substitution at position 20 were not generated because this nucleotide is part of the U6 promoter sequence and therefore must remain a guanine to avoid affecting expression.)

For EGFP target site #2, single mismatches in positions 1-10 of the gRNA have dramatic effects on associated Cas9 activity (FIG. 2C, middle panel), consistent with previous studies that suggest mismatches at the 5′ end of gRNAs are better tolerated than those at the 3′ end (Jiang et al., Nat Biotechnol 31, 233-239 (2013); Cong et al., Science 339, 819-823 (2013); Jinek et al., Science 337, 816-821 (2012)). However, with EGFP target sites #1 and #3, single mismatches at all but a few positions in the gRNA appear to be well tolerated, even within the 3′ end of the sequence. Furthermore, the specific positions that were sensitive to mismatch differed for these two targets (FIG. 2C, compare top and bottom panels)—for example, target site #1 was particularly sensitive to a mismatch at position 2 whereas target site #3 was most sensitive to mismatches at positions 1 and 8.

Example 1b Multiple Mismatches

To test the effects of more than one mismatch at the gRNA/DNA interface, a series of variant gRNAs bearing double Watson-Crick transversion mismatches in adjacent and separated positions were created and the abilities of these gRNAs to direct Cas9 nuclease activity were tested in human cells using the EGFP disruption assay. All three target sites generally showed greater sensitivity to double alterations in which one or both mismatches occur within the 3′ half of the gRNA targeting region. However, the magnitude of these effects exhibited site-specific variation, with target site #2 showing the greatest sensitivity to these double mismatches and target site #1 generally showing the least. To test the number of adjacent mismatches that can be tolerated, variant gRNAs were constructed bearing increasing numbers of mismatched positions ranging from positions 19 to 15 in the 5′ end of the gRNA targeting region (where single and double mismatches appeared to be better tolerated).

Testing of these increasingly mismatched gRNAs revealed that for all three target sites, the introduction of three or more adjacent mismatches results in significant loss of RGN activity. A sudden drop off in activity occurred for three different EGFP-targeted gRNAs as one makes progressive mismatches starting from position 19 in the 5′ end and adding more mismatches moving toward the 3′ end. Specifically, gRNAs containing mismatches at positions 19 and 19+18 show essentially full activity whereas those with mismatches at positions 19+18+17, 19+18+17+16, and 19+18+17+16+15 show essentially no difference relative to a negative control (FIG. 2F). (Note that we did not mismatch position 20 in these variant gRNAs because this position needs to remain as a G because it is part of the U6 promoter that drives expression of the gRNA.)

Additional proof of that shortening gRNA complementarity might lead to RGNs with greater specificities was obtained in the following experiment: for four different EGFP-targeted gRNAs (FIG. 2H), introduction of a double mismatch at positions 18 and 19 did not significantly impact activity. However, introduction of another double mismatch at positions 10 and 11 then into these gRNAs results in near complete loss of activity. Interestingly introduction of only the 10/11 double mismatches does not generally have as great an impact on activity.

Taken together, these results in human cells confirm that the activities of RGNs can be more sensitive to mismatches in the 3′ half of the gRNA targeting sequence. However, the data also clearly reveal that the specificity of RGNs is complex and target site-dependent, with single and double mismatches often well tolerated even when one or more mismatches occur in the 3′ half of the gRNA targeting region. Furthermore, these data also suggest that not all mismatches in the 5′ half of the gRNA/DNA interface are necessarily well tolerated.

In addition, these results strongly suggest that gRNAs bearing shorter regions of complementarity (specifically ˜17 nts) will be more specific in their activities. We note that 17 nts of specificity combined with the 2 nts of specificity conferred by the PAM sequence results in specification of a 19 bp sequence, one of sufficient length to be unique in large complex genomes such as those found in human cells.

Example 1c Off-Target Mutations

To determine whether off-target mutations for RGNs targeted to endogenous human genes could be identified, six single gRNAs that target three different sites in the VEGFA gene, one in the EMX1 gene, one in the RNF2 gene, and one in the FANCF gene were used (Table 1 and Table A). These six gRNAs efficiently directed Cas9-mediated indels at their respective endogenous loci in human U2OS.EGFP cells as detected by T7 Endonuclease I (T7EI) assay (Methods above and Table 1). For each of these six RGNs, we then examined dozens of potential off-target sites (ranging in number from 46 to as many as 64) for evidence of nuclease-induced NHEJ-mediated indel mutations in U2OS.EGFP cells. The loci assessed included all genomic sites that differ by one or two nucleotides as well as subsets of genomic sites that differ by three to six nucleotides and with a bias toward those that had one or more of these mismatches in the 5′ half of the gRNA targeting sequence (Table B). Using the T7EI assay, four off-target sites (out of 53 candidate sites examined) for VEGFA site 1, twelve (out of 46 examined) for VEGFA site 2, seven (out of 64 examined) for VEGFA site 3 and one (out of 46 examined) for the Erna site (Table 1 and Table B) were readily identified. No off-target mutations were detected among the 43 and 50 potential sites examined for the RNF2 or FANCF genes, respectively (Table B). The rates of mutation at verified off-target sites were very high, ranging from 5.6% to 125% (mean of 40%) of the rate observed at the intended target site (Table 1). These bona fide off-targets included sequences with mismatches in the 3′ end of the target site and with as many as a total of five mismatches, with most off-target sites occurring within protein coding genes (Table 1). DNA sequencing of a subset of off-target sites provided additional molecular confirmation that indel mutations occur at the expected RGN cleavage site (FIGS. 8A-C).

TABLE 1 On- and off-target mutations induced by RGNs designed to endogenous human genes SEQ Indel Mutation Frequency Site ID (%) ± SEM Target name Sequence NO: U2OS.EGFP HEK293 K562 Gene Target  T1 GGGTGGGGGGAGTTTGCTCCTGG 1059. 26.0 ± 2.9 10.5 ± 0.07 3.33 ± 0.42 VEGFA 1 OT1-3 GG A TGG A GGGAGTTTGCTCCTGG 1060. 25.7 ± 9.1 18.9 ± 0.77 2.93 ± 0.04 IGDCC3 (VEGFA OT1-4 GGG A GGG T GGAGTTTGCTCCTGG 1061.  9.2 ± 0.8 8.32 ± 0.51 N.D. LOC116437 Site 1) OT1-6 C GG G GG A GGGAGTTTGCTCCTGG 1062.  5.3 ± 0.2 3.67 ± 0.09 N.D. CACNA2D OT1-11 GGG GA GGGG A AGTTTGCTCCTGG 1063. 17.1 ± 4.7 8.54 ± 0.16 N.D. Target  T2 GACCCCCTCCACCCCGCCTCCGG 1064. 50.2 ± 4.9 38.6 ± 1.92 15.0 ± 0.25 VEGFA 2  OT2-1 GACCCCC C CCACCCCGCCCCCGG 1065. 14.4 ± 3.4 33.6 ± 1.17 4.10 ± 0.05 FMN1 (VEGFA OT2-2 G GG CCCCTCCACCCCGCCTCTGG 1066. 20.0 ± 6.2 15.6 ± 0.30 3.00 ± 0.06 PAX6 Site 2) OT2-6 CTA CCCCTCCACCCCGCCTCCGG 1067.  8.2 ± 1.4 15.0 ± 0.64 5.24 ± 0.22 PAPD7 OT2-9 G C CCCC AC CCACCCCGCCTCTGG 1068. 50.7 ± 5.6 30.7 ± 1.44 7.05 ± 0.48 LAMA3 OT2-15 T ACCCCC CA CACCCCGCCTCTGG 1069.  9.7 ± 4.5 6.97 ± 0.10 1.34 ± 0.15 SPNS3 OT2-17 ACA CCCC C CCACCCCGCCTCAGG 1070. 14.0 ± 2.8 12.3 ± 0.45 1.80 ± 0.03 OT2-19 ATT CCCC C CCACCCCGCCTCAGG 1071. 17.0 ± 3.3 19.4 ± 1.35 N.D. HDLBP OT2-20 CC CC A CC C CCACCCCGCCTCAGG 1072.  6.1 ± 1.3 N.D. N.D. ABLIM1 OT2-23 CG CCC T C C CCACCCCGCCTCCGG 1073. 44.4 ± 6.7 28.7 ± 1.15 4.18 ± 0.37 CALY OT2-24 CT CCCC AC CCACCCCGCCTCAGG 1074. 62.8 ± 5.0 29.8 ± 1.08 21.1 ± 1.68 OT2-29 TG CCCC TC CCACCCCGCCTCTGG 1075. 13.8 ± 5.2 N.D. N.D. ACLY OT2-34 AGG CCCC CA CACCCCGCCTCAGG 1076.  2.8 ± 1.5 N.D. N.D. Target  T3 GGTGAGTGAGTGTGTGTGTGAGG 1077. 49.4 ± 3.8 35.7 ± 1.26 27.9 ± 0.52 VEGFA 3  OT3-1 GGTGAGTGAGTGTGTG T GTGAGG 1078.  7.4 ± 3.4 8.97 ± 0.80 N.D. (abParts) (VEGFA OT3-2 A GTGAGTGAGTGTGTG T GTGGGG 1079. 24.3 ± 9.2 23.9 ± 0.08  8.9 ± 0.16 MAX Site 3) OT3-4 G C TGAGTGAGTGT A TGCGTGTGG 1080. 20.9 ± 11.8 11.2 ± 0.23 N.D. OT3-9 GGTGAGTGAGTG C GTGCG G GTGG 1081.  3.2 ± 0.3 2.34 ± 0.21 N.D. TPCN2 OT3-17 G T TGAGTGA A TGTGTGCGTGAGG 1082.  2.9 ± 0.2 1.27 ± 0.02 N.D. SLIT1 OT3-18 T GTG G GTGAGTGTGTGCGTGAGG 1083. 13.4 ± 4.2 12.1 ± 0.24 2.42 ± 0.07 COMDA OT3-20 A G A GAGTGAGTGTGTGC A TGAGG 1084. 16.7 ± 3.5 7.64 ± 0.05 1.18 ± 0.01 Target  T4 GAGTCCGAGCAGAAGAAGAAGGG 1085. 42.1 ± 0.4 26.0 ± 0.70 10.7 ± 0.50 EMX1 4 OT4-1 GAGT TA GAGCAGAAGAAGAAAGG 1086. 16.8 ± 0.2 8.43 ± 1.32 2.54 ± 0.02 HCN1 (EMX1) Target   T5 GTCATCTTAGTCATTACCTGTGG 1087. 26.6 ± 6.0 --- --- RNF2 5 (RNF2) Target  T6 GGAATCCCTTCTGCAGCACCAGG 1088. 33.2 ± 6.5 --- --- FANCF 6  (FANCF) “OT” indicates off-target sites (with numbering of sites as in Table E). Mismatches from the on-target (within the 20 bp region to which the gRNA hybridizes) are highlighted as bold, underlined text. Mean indel mutation frequencies in U2OS.EGFP, HEK293, and K562 cells were determined as described in Methods. Genes in which sites were located (if any) are shown. All sites listed failed to show any evidence of modification in cells transfected with Cas9 expression plasmid and a control U6 promoter plasmid that did not express a functional gRNA. N.D. = none detected; --- = not tested.

Example 1d Off-Target Mutations in Other Cell Types

Having established that RGNs can induce off-target mutations with high frequencies in U2OS.EGFP cells, we next sought to determine whether these nucleases would also have these effects in other types of human cells. We had chosen U2OS.EGFP cells for our initial experiments because we previously used these cells to evaluate the activities of TALENs¹⁵ but human HEK293 and K562 cells have been more widely used to test the activities of targeted nucleases. Therefore, we also assessed the activities of the four RGNs targeted to VEGFA sites 1, 2, and 3 and the EMX1 site in HEK293 and K562 cells. We found that each of these four RGNs efficiently induced NHEJ-mediated indel mutations at their intended on-target site in these two additional human cell lines (as assessed by T7EI assay) (Table 1), albeit with somewhat lower mutation frequencies than those observed in U2OS.EGFP cells. Assessment of the 24 off-target sites for these four RGNs originally identified in U2OS.EGFP cells revealed that many were again mutated in HEK293 and K562 cells with frequencies similar to those at their corresponding on-target site (Table 1). As expected, DNA sequencing of a subset of these off-target sites from HEK293 cells provided additional molecular evidence that alterations are occurring at the expected genomic loci (FIGS. 9A-C). We do not know for certain why in HEK293 cells four and in K562 cells eleven of the off-target sites identified in U2OS.EGFP cells did not show detectable mutations. However, we note that many of these off-target sites also showed relatively lower mutation frequencies in U2OS.EGFP cells. Therefore, we speculate that mutation rates of these sites in HEK293 and K562 cells may be falling below the reliable detection limit of our T7EI assay (˜2-5%) because RGNs generally appear to have lower activities in HEK293 and K562 cells compared with U2OS.EGFP cells in our experiments. Taken together, our results in HEK293 and K562 cells provide evidence that the high-frequency off-target mutations we observe with RGNs will be a general phenomenon seen in multiple human cell types.

Example 1e Titration of gRNA- and Cas9-Expressing Plasmid Amounts Used for the EGFP Disruption Assay

Single gRNAs were generated for three different sequences (EGFP SITES 1-3, shown above) located upstream of EGFP nucleotide 502, a position at which the introduction of frameshift mutations via non-homologous end-joining can robustly disrupt expression of EGFP (Maeder, M. L. et al., Mol Cell 31, 294-301 (2008); Reyon, D. et al., Nat Biotech 30, 460-465 (2012)).

For each of the three target sites, a range of gRNA-expressing plasmid amounts (12.5 to 250 ng) was initially transfected together with 750 ng of a plasmid expressing a codon-optimized version of the Cas9 nuclease into our U2OS.EGFP reporter cells bearing a single copy, constitutively expressed EGFP-PEST reporter gene. All three RGNs efficiently disrupted EGFP expression at the highest concentration of gRNA-encoding plasmid (250 ng) (FIG. 3E (top)). However, RGNs for target sites #1 and #3 exhibited equivalent levels of disruption when lower amounts of gRNA-expressing plasmid were transfected whereas RGN activity at target site #2 dropped immediately when the amount of gRNA-expressing plasmid transfected was decreased (FIG. 3E(top)).

The amount of Cas9-encoding plasmid (range from 50 ng to 750 ng) transfected into our U2OS.EGFP reporter cells was titrated and EGFP disruption assayed. As shown in FIG. 3F (top), target site #1 tolerated a three-fold decrease in the amount of Cas9-encoding plasmid transfected without substantial loss of EGFP disruption activity. However, the activities of RGNs targeting target sites #2 and #3 decreased immediately with a three-fold reduction in the amount of Cas9 plasmid transfected (FIG. 3F (top)). Based on these results, 25 ng/250 ng, 250 ng/750 ng, and 200 ng/750 ng of gRNA-/Cas9-expressing plasmids were used for EGFP target sites #1, #2, and #3, respectively, for the experiments described in Examples 1a-1d.

The reasons why some gRNA/Cas9 combinations work better than others in disrupting EGFP expression is not understood, nor is why some of these combinations are more or less sensitive to the amount of plasmids used for transfection. Although it is possible that the range of off-target sites present in the genome for these three gRNAs might influence each of their activities, no differences were seen in the numbers of genomic sites that differ by one to six bps for each of these particular target sites (Table C) that would account for the differential behavior of the three gRNAs.

TABLE C Numbers of off-target sites in the human genome for six RGNs targeted to endogenous human genes and three RGNs targeted to the EGFP reporter gene Number of mismatches to on-target site Target Site 0 1 2 3 4 5 6 Target 1 (VEGFA Site 1) 1 1 4 32 280 2175 13873 Target 2 (VEGFA Site 2) 1 0 2 35 443 3889 17398 Target 3 (VEGFA Site 3) 1 1 17 377 6028 13398 35517 Target 4 (EMX) 1 0 1 18 276 2309 15731 Target 5 (RNF2) 1 0 0 6 116 976 7443 Target 6 (FANCF) 1 0 1 18 271 1467 9551 EGFP Target Site #1 0 0 3 10 156 1365 9755 EGFP Target Site #2 0 0 0 11 96 974 7353 EGFP Target Site #3 0 0 1 14 165 1439 10361 Off-target sites for each of the six RGNs targeted to the VEGFA, RNF2, FANCF, and EMX1 genes and the three RGNs targeted to EGFP Target Sites #1, #2 and #3 were identified in human genome sequence build GRCh37. Mismatches were only allowed for the 20 nt region to which the gRNA anneals and not to the PAM sequence.

Example 2 Shortening gRNA Complementarity Length to Improve RGN Cleavage Specificity

It was hypothesized that off-target effects of RGNs might be minimized without compromising on-target activity simply by decreasing the length of the gRNA-DNA interface, an approach that at first might seem counterintuitive. Longer gRNAs can actually function less efficiently at the on-target site (see below and Hwang et al., 2013a; Ran et al., 2013). In contrast, as shown above in Example 1, gRNAs bearing multiple mismatches at their 5′ ends could still induce robust cleavage of their target sites (FIGS. 2A and 2C-2F), suggesting that these nucleotides might not be required for full on-target activity. Therefore, it was hypothesized that truncated gRNAs lacking these 5′ nucleotides might show activities comparable to full-length gRNAs (FIG. 2A). It was speculated that if the 5′ nucleotides of full-length gRNAs are not needed for on-target activity then their presence might also compensate for mismatches at other positions along the gRNA-target DNA interface. If this were true, it was hypothesized that gRNAs might have greater sensitivity to mismatches and thus might also induce substantially lower levels of Cas9-mediated off-target mutations (FIG. 2A).

Experimental Procedures

The following experimental procedures were used in Example 2.

Plasmid Construction

All gRNA expression plasmids were assembled by designing, synthesizing, annealing, and cloning pairs of oligonucleotides (IDT) harboring the complementarity region into plasmid pMLM3636 (available from Addgene) as described above (Example 1). The resulting gRNA expression vectors encode a ˜100 nt gRNA whose expression is driven by a human U6 promoter. The sequences of all oligonucleotides used to construct gRNA expression vectors are shown in Table D. The Cas9 D10A nickase expression plasmid (pJDS271) bearing a mutation in the RuvC endonuclease domain was generated by mutating plasmid pJDS246 using a QuikChange kit (Agilent Technologies) with the following primers: Cas9 D10A sense primer 5′-tggataaaaagtattctattggtttagccatcggcactaattccg-3′ (SEQ ID NO:1089); Cas9 D10A antisense primer 5′-cggaattagtgccgatggctaaaccaatagaatactffitatcca-3′ (SEQ ID NO:1090). All the targeted gRNA plasmids and the Cas9 nickase plasmids used in this study are available through the non-profit plasmid distribution service Addgene (addgene.org/crispr-cas).

TABLE D Sequences of oligonucleotides used to construct gRNA expression plasmids EGFP Target Site 1 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1

G C A C G G G C A G C T T G C C G

G C A C G G G C A G C T T G C C

G G C A C G G G C A G C T T G C

G G G C A C G G G C A G C T T G

C G G G C A C G G G C A G C T T

C C G G G C A C G G G C A G C T

G C C G G G C A C G G G C A G C

T G C C G G G C A C G G G C A G

T T G C C G G G C A C G G G C A

C T T G C C G G G C A C G G G C

G C T T G C C G G G C A C G G G

A G C T T G C C G G G C A C G G

C A G C T T G C C G G G C A C G

G C A G C T T G C C G G G C A C

G G C A G C T T G C C G G G C A

G G G C A G C T T G C C G G G C

C G G G C A G C T T G C C G G G

A C G G G C A G C T T G C C G G G C A C G G G C A G C T T G C C

G C A C G G G C A G C T T G

G G G C A C G G G C A G C T

C C G G G C A C G G G C A G

T G C C G G G C A C G G G C

C T T G C C G G G C A C G G

A G C T T G C C G G G C A C

G C A G C T T G C C G G G C

G G G C A G C T T G C C G G G

C G G G C A G C T T G C C G G oligo- SEQ oligo- SEQ SEQ ID nucleotide ID nucleotide ID NO: 1 (5′ to 3′) NO: 2 (5′ to 3′) NO: 2602 ACACCGCACGGGCAGCTTGCCGGG 1091. AAAACGGGGCAAGCTGCCCGTGCG 1180. 2603 ACACCGCACGGGCAGCTTGCCGCG 1092. AAAACGCGGCAAGCTGCCCGTGCG 1181. 2604 ACACCGCACGGGCAGCTTGCCCGG 1093. AAAACCGGGCAAGCTGCCCGTGCG 1182. 2605 ACACCGCACGGGCAGCTTGCGGGG 1094. AAAACCCCGCAAGCTGCCCGTGCG 1183. 2606 ACACCGCACGGGCAGCTTGGCGGG 1095. AAAACCCGCCAAGCTGCCCGTGCG 1184. 2607 ACACCGCACGGGCAGCTTCCCGGG 1096. AAAACCCGGGAAGCTGCCCGTGCG 1185. 2608 ACACCGCACGGGCAGCTAGCCGGG 1097. AAAACCCGGCTAGCTGCCCGTGCG 1186. 2609 ACACCGCACGGGCAGCATGCCGGG 1098. AAAACCCGGCATGCTGCCCGTGCG 1187. 2610 ACACCGCACGGGCAGGTTGCCGGG 1099. AAAACCCGGCAACCTGCCCGTGCG 1188. 2611 ACACCGCACGGGCACCTTGCCGGG 1100. AAAACCCGGCAAGGTGCCCGTGCG 1189. 2612 ACACCGCACGGGCAGCTTGCCGGG 1101. AAAACCCGGCAAGCAGCCCGTGCG 1190. 2613 ACACCGCACGGGGAGCTTGCCGGG 1102. AAAACCCGGCAAGCTCCCCGTGCG 1191. 2614 ACACCGCACGGCCAGCTTGCCGGG 1103. AAAACCCGGCAAGCTGGCCGTGCG 1192. 2615 ACACCGCACGGGCAGCTTGCCGGG 1104. AAAACCCGGCAAGCTGCGCGTGCG 1193. 2616 ACACCGCACCGGCAGCTTGCCGGG 1105. AAAACCCGGCAAGCTGCCGGTGCG 1194. 2617 ACACCGCAGGGGCAGCTTGCCGGG 1106. AAAACCCGGCAAGCTGCCCCTGCG 1195. 2618 ACACCGCTCGGGCAGCTTGCCGGG 1107. AAAACCCGGCAAGCTGCCCGAGCG 1196. 2619 ACACCGGACGGGCAGCTTGCCGGG 1108. AAAACCCGGCAAGCTGCCCGTCCG 1197. 2620 ACACCGCACGGGCAGCTTGCCCCG 1109. AAAACGGGGCAAGCTGCCCGTGCG 1198. 2621 ACACCGCACGGGCAGCTTGGGGGG 1110. AAAACCCCCCAAGCTGCCCGTGCG 1199. 2622 ACACCGCACGGGCAGCTACCCGGG 1111. AAAACCCGGGTAGCTGCCCGTGCG 1200. 2623 ACACCGCACGGGCAGGATGCCGGG 1112. AAAACCCGGCATCCTGCCCGTGCG 1201. 2624 ACACCGCACGGGCTCCTTGCCGGG 1113. AAAACCCGGCAAGGAGCCCGTGCG 1202. 2625 ACACCGCACGGCGAGCTTGCCGGG 1114. AAAACCCGGCAAGCTCGCCGTGCG 1203. 2626 ACACCGCACCCGCAGCTTGCCGGG 1115. AAAACCCGGCAAGCTGCGGGTGCG 1204. 2627 ACACCGCTGGGGCAGCTTGCCGGG 1116. AAAACCCGGCAAGCTGCCCCAGCG 1205. 2628 ACACCGGTCGGGCAGCTTGCCGGG 1117. AAAACCCGGCAAGCTGCCCGACCG 1206. EGFP Target Site 2 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1

G C C G T T C T T C T G C T T G

G C C G T T C T T C T G C T T

T G C C G T T C T T C T G C T

G T G C C G T T C T T C T G C

T G T G C C G T T C T T C T G

T T G T G C C G T T C T T C T

C T T G T G C C G T T C T T C

G C T T G T G C C G T T C T T

T G C T T G T G C C G T T C T

C T G C T T G T G C C G T T C

T C T G C T T G T G C C G T T

T T C T G C T T G T G C C G T

C T T C T G C T T G T G C C G

T C T T C T G C T T G T G C C

T T C T T C T G C T T G T G C

G T T C T T C T G C T T G T G

C G T T C T T C T G C T T G T G C C G T T C T T C T G C T T

G C C G T T C T T C T G C

G T G C C G T T C T T C T

T T G T G C C G T T C T T

G C T T G T G C C G T T C

C T G C T T G T G C C G T

T T C T G C T T G T G C C

T C T T C T G C T T G T G

G T T C T T C T G C T T G T oligo- SEQ oligo- SEQ SEQ ID nucleotide ID nucleotide ID NO: 1 (5′ to 3′) NO: 2 (5′ to 3′) NO: 2629 ACACCGCCGTTCTTCTGCTTGTG 1118. AAAACACAAGCAGAAGAACGGCG 1207. 2630 ACACCGCCGTTCTTCTGCTTGAG 1119. AAAACTCAAGCAGAAGAACGGCG 1208. 2631 ACACCGCCGTTCTTCTGCTTCTG 1120. AAAACAGAAGCAGAAGAACGGCG 1209. 2632 ACACCGCCGTTCTTCTGCTAGTG 1121. AAAACACTAGCAGAAGAACGGCG 1210. 2633 ACACCGCCGTTCTTCTGCATGTG 1122. AAAACACATGCAGAAGAACGGCG 1211. 2634 ACACCGCCGTTCTTCTGGTTGTG 1123. AAAACACAACCAGAAGAACGGCG 1212. 2635 ACACCGCCGTTCTTCTCCTTGTG 1124. AAAACACAAGGAGAAGAACGGCG 1213. 2636 ACACCGCCGTTCTTCAGCTTGTG 1125. AAAACACAAGCTGAAGAACGGCG 1214. 2637 ACACCGCCGTTCTTGTGCTTGTG 1126. AAAACACAAGCACAAGAACGGCG 1215. 2638 ACACCGCCGTTCTACTGCTTGTG 1127. AAAACACAAGCAGTAGAACGGCG 1216. 2639 ACACCGCCGTTCATCTGCTTGTG 1128. AAAACACAAGCAGATGAACGGCG 1217. 2640 ACACCGCCGTTGTTCTGCTTGTG 1129. AAAACACAAGCAGAACAACGGCG 1218. 2641 ACACCGCCGTACTTCTGCTTGTG 1130. AAAACACAAGCAGAAGTACGGCG 1219. 2642 ACACCGCCGATCTTCTGCTTGTG 1131. AAAACACAAGCAGAAGATCGGCG 1220. 2643 ACACCGCCCTTCTTCTGCTTGTG 1132. AAAACACAAGCAGAAGAAGGGCG 1221. 2644 ACACCGCGGTTCTTCTGCTTGTG 1133. AAAACACAAGCAGAAGAACCGCG 1222. 2645 ACACCGGCGTTCTTCTGCTTGTG 1134. AAAACACAAGCAGAAGAACGCCG 1223. 2646 ACACCGCCGTTCTTCTGCTTCAG 1135. AAAACTGAAGCAGAAGAACGGCG 1224. 2647 ACACCGCCGTTCTTCTGCAAGTG 1136. AAAACACTTGCAGAAGAACGGCG 1225. 2648 ACACCGCCGTTCTTCTCGTTGTG 1137. AAAACACAACGAGAAGAACGGCG 1226. 2649 ACACCGCCGTTCTTGAGCTTGTG 1138. AAAACACAAGCTCAAGAACGGCG 1227. 2650 ACACCGCCGTTCAACTGCTTGTG 1139. AAAACACAAGCAGTTGAACGGCG 1228. 2651 ACACCGCCGTAGTTCTGCTTGTG 1140. AAAACACAAGCAGAACTACGGCG 1229. 2652 ACACCGCCCATCTTCTGCTTGTG 1141. AAAACACAAGCAGAAGATGGGCG 1230. 2653 ACACCGGGGTTCTTCTGCTTGTG 1142. AAAACACAAGCAGAAGAACCCCG 1231. EGFP Target Site 3 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1

G G T G C A G A T G A A C T T C

G G T G C A G A T G A A C T T

A G G T G C A G A T G A A C T

C A G G T G C A G A T G A A C

T C A G G T G C A G A T G A A

T T C A G G T G C A G A T G A

C T T C A G G T G C A G A T G

A C T T C A G G T G C A G A T

A A C T T C A G G T G C A G A

G A A C T T C A G G T G C A G

T G A A C T T C A G G T G C A

A T G A A C T T C A G G T G C

G A T G A A C T T C A G G T G

A G A T G A A C T T C A G G T

C A G A T G A A C T T C A G G

G C A G A T G A A C T T C A G

T G C A G A T G A A C T T C A G G T G C A G A T G A A C T T

G G T G C A G A T G A A C

C A G G T G C A G A T G A

T T C A G G T G C A G A T

A C T T C A G G T G C A G

G A A C T T C A G G T G C

A T G A A C T T C A G G T

A G A T G A A C T T C A G

G C A G A T G A A C T T C A oligo- SEQ oligo- SEQ SEQ ID nucleotide ID nucleotide ID NO: 1 (5′ to 3′) NO: 2 (5′ to 3′) NO: 2654 ACACCGGTGCAGATGAACTTCAG 1143. AAAACTCTAGTTCATCTGCACCG 1232. 2655 ACACCGGTGCAGATGAACTTCTG 1144. AAAACTCAAGTTCATCTGCACCG 1233. 2656 ACACCGGTGCAGATGAACTTGAG 1145. AAAACTGTAGTTCATCTGCACCG 1234. 2657 ACACCGGTGCAGATGAACTACAG 1146. AAAACTGATGTTCATCTGCACCG 1235. 2658 ACACCGGTGCAGATGAACATCAG 1147. AAAACTGAACTTCATCTGCACCG 1236. 2659 ACACCGGTGCAGATGAAGTTCAG 1148. AAAACTGAAGATCATCTGCACCG 1237. 2660 ACACCGGTGCAGATGATCTTCAG 1149. AAAACTGAAGTACATCTGCACCG 1238. 2661 ACACCGGTGCAGATGTACTTCAG 1150. AAAACTGAAGTTGATCTGCACCG 1239. 2662 ACACCGGTGCAGATCAACTTCAG 1151. AAAACTGAAGTTCTTCTGCACCG 1240. 2663 ACACCGGTGCAGAAGAACTTCAG 1152. AAAACTGAAGTTCAACTGCACCG 1241. 2664 ACACCGGTGCAGTTGAACTTCAG 1153. AAAACTGAAGTTCATGTGCACCG 1242. 2665 ACACCGGTGCACATGAACTTCAG 1154. AAAACTGAAGTTCATCAGCACCG 1243. 2666 ACACCGGTGCTGATGAACTTCAG 1155. AAAACTGAAGTTCATCTCCACCG 1244. 2667 ACACCGGTGGAGATGAACTTCAG 1156. AAAACTGAAGTTCATCTGGACCG 1245. 2668 ACACCGGTCCAGATGAACTTCAG 1157. AAAACTGAAGTTCATCTGCTCCG 1246. 2669 ACACCGGAGCAGATGAACTTCAG 1158. AAAACTGAAGTTCATCTGCAGCG 1247. 2670 ACACCGCTGCAGATGAACTTCAG 1159. AAAACTGAAGTTCATCTGCAGCG 1248. 2671 ACACCGGTGCAGATGAACTTGTG 1160. AAAACACAAGTTCATCTGCACCG 1249. 2672 ACACCGGTGCAGATGAACAACAG 1161. AAAACTGTTGTTCATCTGCACCG 1250. 2673 ACACCGGTGCAGATGATGTTCAG 1162. AAAACTGAACATCATCTGCACCG 1251. 2674 ACACCGGTGCAGATCTACTTCAG 1163. AAAACTGAAGTAGATCTGCACCG 1252. 2675 ACACCGGTGCAGTAGAACTTCAG 1164. AAAACTGAAGTTCTACTGCACCG 1253. 2676 ACACCGGTGCTCATGAACTTCAG 1165. AAAACTGAAGTTCATGAGCACCG 1254. 2677 ACACCGGTCGAGATGAACTTCAG 1166. AAAACTGAAGTTCATCTCGACCG 1255. 2678 ACACCGCAGCAGATGAACTTCAG 1167. AAAACTGAAGTTCATCTGCTGCG 1256. Endogenous Target 1 (VEGFA Site 1 tru-gRNA): 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 G T G G G G G G A G T T T G C T C C oligo- oligo- SEQ ID nucleotide nucleotide NO: 1 (5′ to 3′) 2 (5′ to 3′) 2679 ACACCGTGGGGGGAGTTTGCTCCG 1168. AAAACGGAGCAAACTCCCCCCACG 1257. Endogenous Target 3 (VEGFA Site 3 tru-gRNA): 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 G A G T G A G T G T G T G C G T G oligo- oligo- SEQ ID nucleotide nucleotide NO: 1 (5′ to 3′) 2 (5′ to 3′) 2680 ACACCGAGTGAGTGTGTGCGTGG 1169. AAAACCACGCACACACTCACTCG 1258. Endogenous Target 4 (EMX1 site 1 tru-gRNA): 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 G T C C G A G C A G A A G A A G A A oligo- oligo- SEQ ID nucleotide nucleotide NO: 1 (5′ to 3′) 2 (5′ to 3′) 2681 ACACCGTCCGAGCAGAAGAAGAAG 1170. AAAACTTCTTCTTCTGCTCGGACG 1259. CTLA full-length gRNA 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 G C A G A T G T A G T G T T T C C A C A oligo- oligo- SEQ ID nucleotide nucleotide NO: 1 (5′ to 3′) 2 (5′ to 3′) 2682 ACACCGCAGATGTAGTGTTTCCACAG 1171. AAAACTGTGGAAACACTACATCTGCG 1260. CTLA tru-gRNA 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 G A T G T A G T G T T T C C A C A oligo- oligo- SEQ ID nucleotide nucleotide NO: 1 (5′ to 3′) 2 (5′ to 3′) 2683 ACACCGATGTAGTGTTTCCACAG 1172. AAAACTGTGGAAACACTACATCG 1261. VEGFA site 4 full-length gRNA 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 T C C C T C T T T A G C C A G A G C C G oligo- oligo- SEQ ID nucleotide nucleotide NO: 1 (5′ to 3′) 2 (5′ to 3′) 2684 ACACCTCCCTCTTTAGCCAGAGCCGG 1173. AAAACCGGCTCTGGCTAAAGAGGGAG 1262. EMX1 site 2 full-length gRNA 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 G C C G T T T G T A C T T T G T C C T C oligo- oligo- SEQ ID nucleotide nucleotide NO: 1 (5′ to 3′) 2 (5′ to 3′) 2685 ACACCGCCGTTTGTACTTTGTCCTCG 1174. AAAACGAGGACAAAGTACAAACGGCG 1263. EMX1 site 2 tru-gRNA 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 G T T T G T A C T T T G T C C T C oligo- oligo- SEQ ID nucleotide nucleotide NO: 1 (5′ to 3′) 2 (5′ to 3′) 2686 ACACCGTTTGTACTTTGTCCTCG 1175. AAAACGAGGACAAAGTACAAACG 1264. EMX1 site 3 full-length gRNA 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 G G G A A G A C T G A G G C T A C A T A oligo- oligo- SEQ ID nucleotide nucleotide NO: 1 (5′ to 3′) 2 (5′ to 3′) 2687 ACACCGGGAAGACTGAGGCTACATAG 1176. AAAACTATGTAGCCTCAGTCTTCCCG 1265. EMX1 site 3 tru-gRNA 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 G A A G A C T G A G G C T A C A T A oligo- SEQ ID oligo-nucleotide nucleotide NO: 1 (5′ to 3′) 2 (5′ to 3′) 2688 ACACCGAAGACTGAGGCTACATAG 1177. AAAACTATGTAGCCTCAGTCTTCG 1266. EMX1 site 4 full-length gRNA 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 G A G G C C C C C A G A G C A G C C A C oligo- SEQ ID oligo-nucleotide nucleotide NO: 1 (5′ to 3′) 2 (5′ to 3′) 2689 ACACCGAGGCCCCCAGAGCAGCCACG 1178. AAAACGTGGCTGCTCTGGGGGCCTCG 1267. EMX1 site 4 tru-gRNA 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 G C C C C C A G A G C A G C C A C oligo- SEQ ID oligo-nucleotide nucleotide NO: 1 (5′ to 3′) 2 (5′ to 3′) 2690 ACACCGCCCCCAGAGCAGCCACG 1179. AAAACGTGGCTGCTCTGGGGGCG 1268.

Human Cell-Based EGFP Disruption Assay

U2OS.EGFP cells harboring a single-copy, integrated EGFP-PEST gene reporter have been previously described (Reyon et al., 2012). These cells were maintained in Advanced DMEM (Life Technologies) supplemented with 10% FBS, 2 mM GlutaMax (Life Technologies), penicillin/streptomycin and 400 μg/ml G418. To assay for disruption of EGFP expression, 2×10⁵ U2OS.EGFP cells were transfected in duplicate with gRNA expression plasmid or an empty U6 promoter plasmid as a negative control, Cas9 expression plasmid (pJDS246) (Example 1 and Fu et al., 2013), and 10 ng of td-Tomato expression plasmid (to control for transfection efficiency) using a LONZA 4D-Nucleofector™, with SE solution and DN100 program according to the manufacturer's instructions. We used 25 ng/250 ng, 250 ng/750 ng, 200 ng/750 ng, and 250 ng/750 ng of gRNA expression plasmid/Cas9 expression plasmid for experiments with EGFP site #1, #2, #3, and #4, respectively. Two days following transfection, cells were trypsinized and resuspended in Dulbecco's modified Eagle medium (DMEM, Invitrogen) supplemented with 10% (vol/vol) fetal bovine serum (FBS) and analyzed on a BD LSRII flow cytometer. For each sample, transfections and flow cytometry measurements were performed in duplicate.

Transfection of Human Cells and Isolation of Genomic DNA

To assess the on-target and off-target indel mutations induced by RGNs targeted to endogenous human genes, plasmids were transfected into U2OS.EGFP or HEK293 cells using the following conditions: U2OS.EGFP cells were transfected using the same conditions as for the EGFP disruption assay described above. HEK293 cells were transfected by seeding them at a density of 1.65×10⁵ cells per well in 24 well plates in Advanced DMEM (Life Technologies) supplemented with 10% FBS and 2 mM GlutaMax (Life Technologies) at 37° C. in a CO₂ incubator. After 22-24 hours of incubation, cells were transfected with 125 ng of gRNA expression plasmid or an empty U6 promoter plasmid (as a negative control), 375 ng of Cas9 expression plasmid (pJDS246) (Example 1 and Fu et al., 2013), and 10 ng of a td-Tomato expression plasmid, using Lipofectamine LTX reagent according to the manufacturer's instructions (Life Technologies). Medium was changed 16 hours after transfection. For both types of cells, genomic DNA was harvested two days post-transfection using an Agencourt DNAdvance genomic DNA isolation kit (Beckman) according to the manufacturer's instructions. For each RGN sample to be assayed, 12 individual 4D transfection replicates were performed, genomic DNA was isolated from each of these 12 transfections, and then these samples were combined to create two “duplicate” pools each consisting of six pooled genomic DNA samples. Indel mutations were then assessed at on-target and off-target sites from these duplicate samples by T7EI assay, Sanger sequencing, and/or deep sequencing as described below.

To assess frequencies of precise alterations introduced by HDR with ssODN donor templates, 2×10⁵ U2OS.EGFP cells were transfected 250 ng of gRNA expression plasmid or an empty U6 promoter plasmid (as a negative control), 750 ng Cas9 expression plasmid (pJDS246), 50 pmol of ssODN donor (or no ssODN for controls), and 10 ng of td-Tomato expression plasmid (as the transfection control). Genomic DNA was purified three days after transfection using Agencourt DNAdvance and assayed for the introduction of a BamHI site at the locus of interest as described below. All of these transfections were performed in duplicate.

For experiments involving Cas9 nickases, 2×10⁵ U2OS.EGFP cells were transfected with 125 ng of each gRNA expression plasmid (if using paired gRNAs) or 250 ng of gRNA expression plasmid (if using a single gRNA), 750 ng of Cas9-D10A nickase expression plasmid (pJDS271), 10 ng of td-Tomato plasmid, and (if performing HDR) 50 pmol of ssODN donor template (encoding the BamHI site). All transfections were performed in duplicate. Genomic DNA harvested two days after transfection (if assaying for indel mutations) or three days after transfection (if assaying for HDR/ssODN-mediated alterations) using the Agencourt DNAdvance genomic DNA isolation kit (Beckman).

T7EI Assays for Quantifying Frequencies of Indel Mutations

T7EI assays were performed as previously described (Example 1 and Fu et al., 2013). In brief, PCR reactions to amplify specific on-target or off-target sites were performed with Phusion high-fidelity DNA polymerase (New England Biolabs) using one of the two following programs: (1) Touchdown PCR program [(98° C., 10 s; 72-62° C., −1° C./cycle, 15 s; 72° C., 30 s)×10 cycles, (98° C., 10 s; 62° C., 15 s; 72° C., 30 s)×25 cycles] or (2) Constant Tm PCR program [(98° C., 10 s; 68° C. or 72° C., 15 s; 72° C., 30 s)×35 cycles], with 3% DMSO or 1 M betaine if necessary. All primers used for these amplifications are listed in Table E. Resulting PCR products ranged in size from 300 to 800 bps and were purified by Ampure XP beads (Agencourt) according to the manufacturer's instructions. 200 ng of purified PCR products were hybridized in 1×NEB buffer 2 in a total volume of 19 μl and denatured to form heteroduplexes using the following conditions: 95° C., 5 minutes; 95 to 85° C., −2° C./s; 85 to 25° C., −0.1° C./s; hold at 4° C. 1 μl of T7 Endonuclease I (New England Biolabs, 10 units/μl) was added to the hybridized PCR products and incubated at 37° C. for 15 minutes. The T7EI reaction was stopped by adding 2 μl of 0.25 M EDTA solution and the reaction products were purified using AMPure XP beads (Agencourt) with elution in 20 μl 0.1×EB buffer (QIAgen). Reactions products were then analyzed on a QIAXCEL capillary electrophoresis system and the frequencies of indel mutations were calculated using the same formula as previously described (Reyon et al., 2012).

TABLE E Mis- mat- ches in non- target Actual Wat- Wat- Pub- Expected com- Target son- son- li- Off-Target pared in Crick Crick ca- Sequences SEQ to on- U2OS. Forward SEQ Reverse SEQ PCR Trans- Trans- Tran- tion- (Expected)- ID target EGFP PCR ID PCR ID Condi- ver- ver- si- ID HS GRCh37 NO: site cells Primer NO: Primer NO: tions sions sions tions Tar- GGGTGGGGGGAG 1269. 0 TCCAGAT 1270. AGGGAGCA 1271. DMSO get TTTGCTCCTGG GGCACA GGAAAGTG 1 TTGTCAG AGGT OT1- GGGTGGGGGGAG 1272. 1 GGGGCCC 1273. ACCCAGAC 1274. No 0 0 1 1 TTTGCCCCAGG ACTCTT TCCTGGTG DMSO CTTCCAT TGGC OT1- GCGTGGGGGGTG 1275. 2 GCTAAGC 1276. ACCACCCT 1277. DMSO 2 0 0 2 TTTGCTCCTGG AGAGAT TTCCCCCA GCCTATG GAAA CC OT1- GGATGGAGGGAG 1278. 2 ACCCCA 1279. GAATCACT 1280. DMSO 0 0 2 3 TTTGCTCCTGG CAGCCAG GCACCTGG GTTTTCA CCATC OT1- GGGAGGGTGGAG 1281. 2 TGCGGCA 1282. TAAAGGGC 1283. DMSO 1 1 0 4 TTTGCTCCTGG ACTTCA GTGCTGGG GACAACC AGAG OT1- GGGTGGGTGGAG 1284. 2 GCATGTC 1285. TGCAGGGC 1286. DMSO 0 2 0 5 TTTGCTACTGG AGGATC CATCTTGT TGACCCC GTGT OT1- CGGGGGAGGGAG 1287. 3 CCACCAC 1288. CTGGGTCT 1289. DMSO 1 1 1 6 TTTGCTCCTGG ATGTTC GTTCCCTG TGGGTGC TGGG OT1- GAGTGGGTGGAG 1290. 3 GGCTCTC 1291. GCAGGTCA 1292. DMSO 0 2 1 7 TTTGCTACAGG CCTGCC AGTTGGAA CTAGTTT CCCG OT1- GGGAGGGGAGAG 1293. 3 GGGGCTG 1294. AGATTTGT 1295. DMSO 1 0 2 8 TTTGTTCCAGG AGAACA GCACTGCC CATGAGA TGCCT TGCA OT1- GGGAGGGGGCAG 1296. 3 CCCGACC 1297. GGACCTCT 1298. DMSO 2 1 0 9 GTTGCTCCAGG TCCGCT GCACACCC CCAAAGC TGGC OT1- GGGAGGGGGGAG 1299. 3 TGCAAGG 1300. CAGGAGGG 1301. DMSO 1 1 1 10 TGTGTTCCGGG TCGCAT GGAAGTGT AGTCCCA GTCC OT1- GGGGAGGGGAAG 1302. 3 GCCCATT 1303. GAGAGCAA 1304. DMSO 0 1 2 11 TTTGCTGCTGG CTTTTT GTTTGTTC GCAGTGG CCCAGG A OT1- GGGGGTGGGGAC 1305. 3 GCCCCCA 1306. GCTGCTGG 1307. DMSO 1 2 0 12 TTTGCTCCAGG GCCCCT TAGGGGAG CTGTTTC CTGG OT1- GGGTCGGGGGAG 1308. 3 CGGCTGC 1309. GGGTGACG 1310. 72C 1 2 0 13 TGGGCTCCAGG CTTCCC CTTGCCAT An- TGAGTCC GAGC neal, 3% DMSO OT1- GGGTGGCTGGAG 1311. 3 TGACCCT 1312. GCTGAGAC 1313. 72C 2 1 0 14 TTTGCTGCTGG GGAGTA AACCAGCC An- CAAAATG CAGCT neal, TTCCCA 3% DMSO OT1- GGGTGGGGGGTG 1314. 3 TGCCTCC 1315. GCAGCCGA 1316. DMSO 1 0 2 15 CCTGCTCCAGG ACCCTT TCCACACT AGCCCCT GGGG OT1- GGTTGAGGGGAG 1317. 3 AACTCAG 1318. CCCAGGAG 1319. DMSO 0 1 2 16 TCTGCTCCAGG GACAAC CAGGGTAC ACTGCCT AATGC GT OT1- GTGTGGGTGGCG 1320. 3 TCCTCCT 1321. CCTTGGAA 1322. DMSO 0 3 0 17 TTTGCTCCAGG TGGAGA GGGGCCTT GGGGCCC GGTGG OT1- AGGTGGTGGGAG 1323. 4 CCGAGGG 1324. GGCTGCTG 1325. DMSO 0 1 3 18 CTTGTTCCTGG CATGGG CGAGTTGC CAATCCT CAAC OT1- AGTTTGGGGGAG 1326. 4 TGCTTTG 1327. GGGTTGCT 1328. DMSO 0 2 2 19 TTTGCCCCAGG CATGGG TGCCCTCT GTCTCAG GTGT ACA OT1- ATGTGTGGGGAA 1329. 4 AGCTCCT 1330. CACAGAAG 1331. DMSO 0 2 2 20 TTTGCTCCAGG TCTCAT GATGTGTG TTCTCTT CAGGTT CTGCTG T OT1- CAGTGGGGGGAG 1332. 4 AGCAGAC 1333. GGTCAGGT 1334. DMSO 1 1 2 21 CTTTCTCCTGG ACAGGT GTGCTGCT GAATGCT AGGCA GCT OT1- GAGGGGGAGCAG 1335. 4 CCTGTGG 1336. ACTGCCTG 1337. No 1 1 2 22 TTTGCTCCAGG GGCTCT CCAAAGTG DMSO CAGGTGC GGTGT TD OT1- GGAGGAGGGGAG 1338. 4 AGCTGCA 1339. TGCCGGGT 1340. DMSO 0 1 3 23 TCTGCTCCAGG CTGGGG AATAGCTG AATGAGT GCTT OT1- GGAGGGGGGGCT 1341. 4 CCAGCCT 1342. GGGGGCTT 1343. 72C 0 3 1 24 TTTGCTCCAGG GGGCAA CCAGGTCA An- CAAAGCG CAGG neal, 3% DMSO, 6% DMSO OT1- GGGCAAGGGGAG 1344. 4 TACCCCC 1345. ACAGGTCC 1346. DMSO 0 1 3 25 GTTGCTCCTGG ACTGCC ATGCTTAG CCATTGC CAGAGGG OT1- GGGTGATTGAAG 1347. 4 GGGTGAT ACGGATT 1348. CCGAGTCC 1349. DMSO 0/ 2 2 26 TTTGCTCCAGG TGAAGTT  CACGAC GTGGCAGA 1 TGCTCCA  GGAGGTG GAGC GG C (SEQ  ID NO:  2225) GGGTGAT TGAAGTT TGCGGCA  GG (SEQ   ID NO:  2226) OT1- GGGTGTGGGGTC 1350. 4 TGTGGTT 1351. TGGCCCAA 1352. DMSO 3 1 0 27 ATTGCTCCAGG GAAGTA TTGGAAGT GGGGACA GATTTCGT GGT OT1- GGTGGGGGTGGG 1353. 4 TGGGATG 1354. GGCCCAAT 1355. DMSO 0 3 1 28 TTTGCTCCAGG GCAGAG CGGTAGAG TCATCAA GATGCA CGT OT1- GTGGGGGTAGAG 1356. 4 ATGGGGC 1357. TGCACCCA 1358. DMSO 0 3 1 29 TTTGCTCCAGG GCTCCA CACAGCCA GTCTGTG GCAA OT1- TAGTGGAGGGAG 1359. 4 GGGGAGG 1360. AATTAGCT 1361. 72C 0 1 3 30 CTTGCTCCTGG GAGGAC GGGCGCGG An- CAGGGAA TGGT neal, 3% DMSO OT1- TGCTCGGGGGAG 1362. 4 ATCCCGT 1363. CAGGCGGC 1364. DMSO 3 1 0 31 TTTGCACCAGG GCAGGA CCCTTGAG AGTCGCC GAAT OT1- TGGAGAGGGGAG 1365. 4 CCCCAAC 1366. TGAGGAGA 1367. DMSO 1 2 1 32 TTGGCTCCTGG CCTTTG ACACCACA CTCAGCG GGCAGA OT1- TGGTGTTGGGAG 1368. 4 ATCGACG 1369. CCCCTCAC 1370. DMSO 0 3 1 33 TCTGCTCCAGG AGGAGG TCAAGCAG GGGCCTT GCCC OT1- TTGGGGGGGCAG 1371. 4 TGCTCAA 1372. CAGGGGCA 1373. No 1 3 0 34 TTTGCTCCTGG GGGGCC GTGGCAGG DMSO TGTTCCA AGTC OT1- AAGTAAGGGAAG 1374. 5 TGCCTGG 1375. GGGAAGGG 1376. DMSO 0 0 5 35 TTTGCTCCTGG CACGCA GGAACAGG GTAGGTG TGCA OT1- AGAAGAGGGGAT 1377. 5 Not  1 1 3 36 TTTGCTCCTGG opti- mized OT1- ATCTGGGGTGAT 1378. 5 ACCTGGG 1379. GCTGCTCG 1380. DMSO 1 3 1 37 TTTGCTCCTGG CTTGCC CAGTTAAG ACTAGGG CACCA OT1- CTCTGCTGGGAG 1381. 5 GTGGCCG 1382. GGTTCCAC 1383. DMSO 3 2 0 38 TTTGCTCCTGG GGCTAC AAGCTGGG TGCTACC GGCA OT1- CTGGTGGGGGAG 1384. 5 Not  1 3 1 39 CTTGCTCCAGG opti- mized OT1- CTTTCGGGGGAG 1385. 5 GCAAGAG 1386. AGAGTCAT 1387. DMSO 2 3 0 40 TTTGCGCCGGG GCGGAG CCATTTCC GAGACCC TGGGGGC OT1- CTTTGGGGTTAG 1388. 5 GGGGTCA 1389. AGGGAATC 1390. 1M 1 4 0 41 TTTGCTCCTGG GTGGTG CTTTTTCC be- ATATCCC ATTGCTTG taine, CCT TTT TD OT1- GCTCTGGGGTAG 1391. 5 AGAGAGG 1392. GCCTCCCC 1393. DMSO 1 3 1 42 TTTGCTCCAGG CCACGT TCCTCCTT GGAGGGT CCCA OT1- GTCTCTCGGGAG 1394. 5 GACAGTG 1395. TCTGACCG 1396. DMSO 3 2 0 43 TTTGCTCCTGG CCTTGC GTATGCCT GATGCAC GACG OT1- TCCTGAGGGCAG 1397. 5 TGTGTGA 1398. TGGTCTAG 1399. DMSO 3 1 1 44 TTTGCTCCAGG ACGCAG TACTTCCT CCTGGCT CCAGCCTT OT1- TCTTTGGGAGAG 1400. 5 GGTTCTC 1401. CCCACTGC 1402. DMSO 1 3 1 45 TTTGCTCCAGG CCTTGG TCCTAGCC CTCCTGT CTGC GA OT1- ACAACTGGGGAG 1403. 6 TGAAGTC 1404. AGCTTTGG 1405. DMSO 3 1 2 46 TTTGCTCCTGG AACAAT TAGTTGGA CTAAGCT GTCTTTGA TCCACC AGG T OT1- ACAAGGTGGAAG 1406. 6 TGATTGG 1407. GCACAGCC 1408. DMSO 2 1 3 47 TTTGCTCCTGG GCTGCA TGCCCTTG GTTCATG GAAG TACA OT1- ACATAGAAGGAG 1409. 6 TCCATGG 1410. AGCGGCTT 1411. DMSO 1 0 5 48 TTTGCTCCAGG GCCCCT CTGCTTCT CTGAAAG GCGA A OT1- AGACCCAGGGAG 1412. 6 GCGGTTG 1413. GAGTTCCT 1414. DMSO 2 0 4 49 TTTGCTCCCGG GTGGGG CCTCCCGC TTGATGC CAGT OT1- AGACCCAGGGAG 1415. 6 AGGCAAG 1416. GCTTTTGC 1417. DMSO 2 0 4 50 TTTGCTCCCGG ATTTTC CTGGGACT CAGTGTG CCGC CAAGA OT1- CACGGAGGGGTG 1418. 6 GCTGCTG 1419. GCTCTGTC 1420. No 3 1 2 51 TTTGCTCCTGG GTCGGG CCACTTCC DMSO CTCTCTG CCTGG TD OT1- CAGAGCTTGGAG 1421. 6 GCTGCGA 1422. CGCCCCTA 1423. DMSO 3 2 1 52 TTTGCTCCAGG GGCTTC GAGCTAAG CGTGAGA GGGGT OT1- CTATTGATGGAG 1424. 6 CCAGGAG 1425. AGGGCTAG 1426. DMSO 1 3 2 53 TTTGCTCCTGG CCTGAG GACTGCAG AGCTGCC TGAGC OT1- CTTTCTAGGGAG 1427. 6 CTGTGCT 1428. GCCTGGGG 1429. DMSO 2 3 1 54 TTTGCTCCTGG CAGCCT CTGTGAGT GGGTGCT AGTTT OT1- GCCATGCTGGAG 1430. 6 AGCTCGC 1431. ACTTGGCA 1432. 72C 4 2 0 55 TTTGCTCCAGG GCCAGA GGCTGAGG An- TCTGTGG CAGG neal, 3% DMSO 1433. 1434. 1435. Tar-  GACCCCCTCCAC 1436. 0 AGAGAAG 1437. CAGCAGAA 1438. DMSO get CCCGCCTCCGG TCGAGG AGTTCATG 2 AAGAGAG GTTTCG AG OT2- GACCCCCCCCAC 1439. 2 TGGACAG 1440. ACTGATCG 1441. DMSO 0 0 2 1 CCCGCCCCCGG CTGCAG ATGATGGC TACTCCC CTATGGGT TG OT2- GGGCCCCTCCAC 1442. 2 CAAGATG 1443. GCAGCCTA 1444. DMSO 1 0 1 2 CCCGCCTCTGG TGCACT TTGTCTCC TGGGCTA TGGT OT2- AACCCCATCCAC 1445. 3 GTCCAGT 1446. AGCATCAT 1447. DMSO 1 1 1 3 CCGGCCTCAGG GCCTGA GCCTCCAG CCCTGGC CTTCA OT2- CACCCCCTCAAC 1448. 3 GCTCCCG 1449. GCAGCTCC 1450. DMSO 1 2 0 4 ACCGCCTCAGG ATCCTC CACCACCC TGCCACC TCAG OT2- CACCCCCTCCCC 1451. 3 GGGGAC 1452. GTGCGTGT 1453. DMSO 1 1 1 5 TCCGCCTCAGG AGGCAGG CCGTTCAC CAAGGAG CCCT OT2- CTACCCCTCCAC 1454. 3 AAGGGGC 1455. CGTGATTC 1456. DMSO 2 1 0 6 CCCGCCTCCGG TGCTGG GAGTTCCT GTAGGAC GGCA OT2- GACCCGCCCCGC 1457. 3 GACCCTC 1458. CTGCGAGA 1459. 1M 1 0 2 7 CCCGCCTCTGG AGGAAG TGCCCCAA be- CTGGGAG ATCG taine, TD OT2- GATCGACTCCAC 1460. 3 CCGCGGC 1461. TGCTGGGA 1462. DMSO 1 1 1 8 CCCGCCTCTGG GCTCTG TTACAGGC CTAGA GCGA OT2- GCCCCCACCCAC 1463. 3 CCAGGTG 1464. TGCCTGGC 1465. DMSO 0 2 1 9 CCCGCCTCTGG GTGTCA CCTCTCTG GCGGAGG AGTCT OT2- GCCCCGCTCCTC 1466. 3 CGACTCC 1467. CAGCGCAG 1468. 1M 2 1 0 10 CCCGCCTCCGG ACGGCG TCCAGCCC be- TCTCAGG GATG taine, TD OT2- GGCCCCCTCCAC 1469. 3 CTTCCCT 1470. GCTACAGG 1471. DMSO 1 1 1 11 CAGGCCTCAGG CCCCCA TTGCACAG GCACCAC TGAGAGGT OT2- GGCCCCCTCCTC 1472. 3 CCCCGGG 1473. CCCAGCCG 1474. 72C 1 0 2 12 CTCGCCTCTGG GAGTCT TTCCAGGT An- GTCCTGA CTTCC neal, 3% DMSO OT2- GGCGCCCTCCAC 1475. 3 GAAGCGC 1476. TCCAGGGT 1477. DMSO 1 0 2 13 CCTGCCTCGGG GAAAAC CCTTCTCG CCGGCTC GCCC OT2- GTCCTCCACCAC 1478. 3 AGGGTGG 1479. CATGGGGC 1480. DMSO 2 0 1 14 CCCGCCTCTGG TCAGGG TCGGACCT AGGCCTT CGTC OT2- TACCCCCCACAC 1481. 3 GGGAAGA 1482. TGCCAGGA 1483. 72C 0 2 1 15 CCCGCCTCTGG GGCAGG AGGAAGCT An- GCTGTCG GGCC neal, 3% DMSO OT2- AACCCATTCCAC 1484. 4 GAGTGAC 1485. CCCTTAGC 1486. 68C 0 1 3 16 CCTGCCTCAGG GATGAG TGCAGTCG An- CCCCGGG CCCC neal, 3% DMSO OT2- ACACCCCCCCAC 1487. 4 CCCATGA 1488. TGAAGATG 1489. DMSO 0 2 2 17 CCCGCCTCAGG GGGGTT GGCAGTTT TGAGTGC GGGG OT2- AGCCCCCACCTC 1490. 4 CACCTGG 1491. ACTGGGGT 1492. DMSO 2 0 2 18 CCCGCCTCTGG GGCATC TGGGGAGG TGGGTGG GGAT OT2- ATTCCCCCCCAC 1493. 4 TCATGAT 1494. CCATTTGT 1495. DMSO 1 0 3 19 CCCGCCTCAGG CCCCAA GCTGATCT AAGGGCT GTGGGT OT2- CCCCACCCCCAC 1496. 4 TGGTGCC 1497. AGGAAATG 1498. DMSO 1 2 1 20 CCCGCCTCAGG CAGAAT TGTTGTGC AGTGGCC CAGGGC A OT2- CCCCCCCACCAC 1499. 4 GCCTCAG 1500. GCCAAGTG 1501. No 2 1 1 21 CCCGCCCCGGG ACAACC TTACTCAT DMSO CTGCCCC CAAGAAAG TD TGG OT2- CCCCCCCCCCCC 1502. 4 GCCGGGA 1503. TCCCGAAC 1504. DMSO 1 2 1 22 CCCGCCTCAGG CAAGAC TCCCGCAA TGAGTTG AACG GG OT2- CGCCCTCCCCAC 1505. 4 TGCTGCA 1506. CTGGAACC 1507. No 1 0 3 23 CCCGCCTCTGG GGTGGT GCATCCTC DMSO TCCGGAG CGCA TD OT2- CTCCCCACCCAC 1508. 4 ACACTGG 1509. GGCTGTGC 1510. DMSO 2 1 1 24 CCCGCCTCAGG TCCAGG CTTCCGAT TCCCGTC GGAA T OT2- CTCTCCCCCCAC 1511. 4 CTCTCCC ATCGCGC 1512. AGGCTTCT 1513. DMSO 3 0 2 25 CCCGCCTCTGG CCCACCC  CCAAAG GGAAAAGT CCCCTCT  CACAGGT CCTCAATG GG CA (SEQ  ID NO:  2227) OT2- GCCTCTCTGCAC 1514. 4 Not  1 1 2 26 CCCGCCTCAGG opti- mized OT2- GTCACTCCCCAC 1515. 4 CCCTCAT 1516. AGCCACAC 1517. DMSO 1 1 2 27 CCCGCCTCTGG GGTGGT ATCTTTCT CTTACGG GGTAGGG CA OT2- TGCCCCCTCCCC 1518. 4 TGCGTCG 1519. AGGGTGGG 1520. DMSO 0 3 1 28 CCAGCCTCTGG CTCATG GTGTACTG CTGGGAG GCTCA OT2- TGCCCCTCCCAC 1521. 4 GAGCTGA 1522. TGGCCTTG 1523. 1M 0 1 3 29 CCCGCCTCTGG GACGGC AACTCTTG be- ACCACTG GGCT taine, TD OT2- TTCCCCTTCCAC 1524. 4 Not  1 2 1 30 CCAGCCTCTGG opti- mized OT2- TTCTCCCTCCTC 1525. 4 AGTGAGA 1526. CAGTAGGT 1527. DMSO 2 1 1 31 CCCGCCTCGGG GTGGCA GGTCCCTT CGAACCA CCGC OT2- ACCCTCGCCCAC 1528. 5 Not  1 1 3 32 CCCGCCTCAGG opti- mized OT2- AGCCAACCCCAC 1529. 5 GGGAGAA 1530. AAGCCGAA 1531. DMSO 0 2 3 33 CCCGCCTCTGG CCTTGT AAGCTGGG CCAGCCT CAAA OT2- AGGCCCCCACAC 1532. 5 CTTCCCA 1533. ACACAGTC 1534. DMSO 1 1 3 34 CCCGCCTCAGG GTGTGG AGAGCTCC CCCGTCC GCCG OT2- AGGCCCCCCCGC 1535. 5 Not  1 0 4 35 CCCGCCTCAGG opti- mized OT2- ATCTGCCACCAC 1536. 5 CTGAGAG 1537. TCGACTGG 1538. 68C 3 0 2 36 CCCGCCTCGGG GGGGAG TCTTGTCC An- GGGGAGG TCCCA neal, 3% DMSO OT2- CATCTTCCCCAC 1539. 5 CAGCCTG 1540. TGCAGCCA 1541. 1M 1 0 4 37 CCCGCCTCTGG CTGCAT AGAGAAAA  be- CGGAAAA AGCCT taine, TD OT2- CTTTCCCTCCAC 1542. 5 TCCCTCT 1543. ACCCGACT 1544. DMSO 2 1 2 38 CCAGCCTCTGG GACCCG TCCTCCCC GAACCCA ATTGC OT2- GTCGAGGTCCAC 1545. 5 TGGGGGT 1546. GCCAGGAG 1547. DMSO 4 1 0 39 CCCGCCTCAGG TGCGTG GACACCAG CTTGTCA GACC OT2- GTCGAGGTCCAC 1548. 5 ATCAGGT 1549. GGCCTGAG 1550. DMSO 4 1 0 40 CCCGCCTCAGG GCCAGG AGTGGAGA AGGACAC GTGG OT2- TCAGACCTCCAC 1551. 5 Not  1 4 0 41 CCCGCCTCAGG opti- mized OT2- TGCAACCTCCTC 1552. 5 TGAGCCA 1553. ACCTCTCC 1554. DMSO 1 3 1 42 CCCGCCTCGGG CATGAA AAGTCTCA TCAAGGC GTAACTCT CTCC CT OT2- ACCAGTCTGCAC 1555. 6 GGTCCCT 1556. CTTTGGTG 1557. DMSO 2 2 2 43 CCCGCCTCTGG CTGTGC GACCTGCA AGTGGAA CAGC OT2- ACTACCCACCTC 1558. 6 GCGAGGC 1559. GCTGGGAC 1560. DMSO 2 2 2 44 CCCGCCTCAGG TGCTGA TACAGACA CTTCCCT TGTGCCA OT2- ATTTCCCCCCCC 1561. 6 ATTTCCT ATTGCAG 1562. AAATCCTG 1563. DMSO 1 1 5 45 CCCGCCTCAGG CCCCCCC GCGTGT CATGGTGA C-  CCAGGCA TGGGAGT CCTCAGG (SEQ   ID NO:  2228) OT2- CCACCATCCCAC 1564. 6 TGCTCTG 1565. ACAGCCTC 1566. DMSO 1 3 2 46 CCCGCCTCTGG CCATTT TTCTCCAT ATGTCCT GACTGAGC ATGAAC T OT2- CCCAAGCCCCAC 1567. 6 TCCGCCC 1568. GCGGTGGG 1569. DMSO 2 3 1 47 CCCGCCTCGGG AAACAG GAAGCCAT GAGGCAG TGAG OT2- CCGCGCTTCCGC 1570. 6 GGGGGTC 1571. CCTGTCGG 1572. DMSO 3 1 2 48 CCCGCCTCTGG TGGCTC GAGAGTGC ACCTGGA CTGC OT2- CCTGCCATGCAC 1573. 6 TCCTGGT 1574. ACTCCAGA 1575. DMSO 3 2 1 49 CCCGCCTCAGG TCATTT TGCAACCA GCTAGAA GGGCT CTCTGG A OT2- CTGCCTCCTCAC 1576. 6 CGTGTGG 1577. GCTTCACC 1578. DMSO 3 0 3 50 CCCGCCTCAGG TGAGCC GTAGAGGC TGAGTCT TGCT OT2- TCTTCTTTCCAC 1579. 6 AGGCCCT 1580. TCAGTGAC 1581. DMSO 0 2 4 51 CCCGCCTCAGG GATAAT AACCTTTT TCATGCT GTATTCGG ACCAA CA OT2- TTGACCCCCCGC 1582. 6 Not  2 2 2 52 CCCGCCTCAGG opti- mized Tar-  GGTGAGTGAGTG 1583. 0 TCCAGAT 1584. AGGGAGCA 1585. DMSO get TGTGCGTGTGG GGCACA GGAAAGTG 3 TTGTCAG AGGT OT3- GGTGAGTGAGTG 1586. 1 GCAGGCA 1587. CACCGACA 1588. DMSO 0 0 1 1 TGTGTGTGAGG AGCTGT CACCCACT CAAGGGT CACC OT3- AGTGAGTGAGTG 1589. 2 GAGGGGG 1590. TACCCGGG 1591. DMSO 0 0 2 2 TGTGTGTGTGG AAGTCA CCGTCTGT CCGACAA TAGA OT3- AGTGTGTGAGTG 1592. 2 GACACCC 1593. TGAATCCC 1594. DMSO 1 0 1 3 TGTGCGTGTGG CACACA TTCACCCC CTCTCAT CAAG GC OT3- GCTGAGTGAGTG 1595. 2 TCCTTTG 1596. CCAATCCA 1597. DMSO 1 0 1 4 TATGCGTGTGG AGGTTC GGATGATT ATCCCCC CCGC OT3- GGTGAGTCAGTG 1598. 2 CAGGGCC 1599. GGGAGGTA 1600. DMSO 1 1 0 5 TGTGAGTGAGG AGGAAC TGTGCGGG ACAGGAA AGTG OT3- GGTGAGTGAGAG 1601. 2 TGCAGCC 1602. GCCCAGGT 1603. DMSO 1 0 1 6 TGTGTGTGTGG TGAGTG GCTAAGCC AGCAAGT CCTC GT OT3- GGTGAGTGAGTG 1604. 2 TACAGCC 1605. TGTGTCAT 1606. 1M 1 1 0 7 AGTGAGTGAGG TGGGTG GGACTTTC be- ATGGAGC CCATTGT taine, TD OT3- GGTGAGTGAGTG 1607. 2 GGCAGGC 1608. TCTCCCCC 1609. DMSO 1 1 0 8 AGTGAGTGAGG ATTAAA AAGGTATC CTCATCA AGAGAGCT GGTCC OT3- GGTGAGTGAGTG 1610. 2 GGGCCTC 1611. GCTGCCGT 1612. DMSO 0 1 1 9 CGTGCGGGTGG CCTGCT CCGAACCC GGTTCTC AAGA OT3- GGTGAGTGTGTG 1613. 2 ACAAACG 1614. ACTCCGAA 1615. DMSO 1 1 0 10 TGTGAGTGTGG CAGGTG AATGCCCC GACCGAA GCAGT OT3- GGTGAGTGTGTG 1616. 2 AGGGGAG 1617. TTGAGAGG 1618. DMSO 1 0 1 11 TGTGCATGTGG GGGACA GTTCAGTG TTGCCT GTTGC OT3- GGTGTGTGAGTG 1619. 2 CTAATGC 1620. AGCCAACG 1621. DMSO 1 0 1 12 TGTGTGTGTGG TTACGG GCAGATGC CTGCGGG AAAT OT3- GGTGTGTGTGTG 1622. 2 GAGCGAA 1623. CACACATG 1624. 68C, 2 0 0 13 TGTGCGTGCGG GTTAAC CACATGCC 3% CCACCGC CCTG DMSO OT3- GGTGTGTGTGTG 1625. 2 GCATGTG 1626. TCCCCCAT 1627. DMSO 2 0 0 14 TGTGCGTGTGG TCTAAC ATCAACAC TGGAGAC ACACA AATAGC A OT3- GGTGTGTGTGTG 1628. 2 GCCCCTC 1629. TGGGCAAA 1630. DMSO 2 0 0 15 TGTGCGTGTGG CCGCCT GGACATGA TTTGTGT AACAGACA OT3- GGTGTGTGTGTG 1631. 2 GCCTCAG 1632. ACGAACAG 1633. DMSO 2 0 0 16 TGTGCGTGTGG CTCTGC ATCATTTT TCTTAAG TCATGGCT CCC TCC OT3- GTTGAGTGAATG 1634. 2 CTCCAGA 1635. CCCTCTCC 1636. DMSO 0 1 1 17 TGTGCGTGAGG GCCTGG GGAAGTGC CCTACCA CTTG OT3- TGTGGGTGAGTG 1637. 2 TCTGTCA 1638. GTTGCCTG 1639. DMSO 0 1 1 18 TGTGCGTGAGG CCACAC GGGATGGG AGTTACC GTAT ACC OT3- ACTGTGTGAGTG 1640. 3 GGGGACC 1641. GGGCATCA 1642. DMSO 2 0 1 19 TGTGCGTGAGG CTCAAG AAGGATGG AGGCACT GGAT OT3- AGAGAGTGAGTG 1643. 3 TGTGGAG 1644. ACAGTGAG 1645. DMSO 1 0 2 20 TGTGCATGAGG GGTGGG GTGCGGTC ACCTGGT TTTGGG OT3- AGCGAGTGGGTG 1646. 3 CGGGGTG 1647. GGTGCAGT 1648. DMSO 0 0 3 21 TGTGCGTGCGG GCAGTG CCAAGAGC ACGTCAA CCCC OT3- AGGGAGTGACTG 1649. 3 AGCTGAG 1650. GGGAGACA 1651. DMSO 1 1 1 22 TGTGCGTGTGG GCAGAG GAGCAGCG TCCCCGA CCTC OT3- AGTGAGTGAGTG 1652. 3 ACCACCA 1653. AGGACGAC 1654. 72C 1 1 1 23 AGTGAGTGAGG GACCCC TTGTGCCC An- ACCTCCA CATTCA neal, 3% DMSO OT3- CATGAGTGAGTG 1655. 3 GGGTCAG 1656. TCCACCCA 1657. 72C 2 0 1 24 TGTGGGTGGGG GACGCA CCCACCCA An- GGTCAGA TCCT neal, 3% DMSO OT3- CGTGAGTGTGTG 1658. 3 ACACTCT 1659. GCCCCCTC 1660. DMSO 2 0 1 25 TATGCGTGTGG GGGCTA ACCACATG GGTGCTG ATGCT GA OT3- GGACTGTGAGTG 1661. 3 GGGGCCA 1662. TGGGGATC 1663. DMSO 3 0 0 26 TGTGCGTGAGG TTCCTC CTTGCTCA TGCTGCA TGGC OT3- GGTGTGTGCCTG 1664. 3 ACACACT 1665. CCTGCACG 1666. DMSO 2 1 0 27 TGTGCGTGTGG GGCTCG AGGCCAGG CATTCAC TGTT CA OT3- GTTTCATGAGTG 1667. 3 TGGGCAC 1668. CTCGCCGC 1669. DMSO 0 3 1 28 TGTGCGTGGGG GTAGTA CGTGACTG AACTGCA TAGG CCA OT3- TGAGTGTGAGTG 1670. 3 TCAGCTG 1671. AGAGCACT 1672. DMSO 2 1 0 29 TGTGCGTGGGG GTCCTG GGGTAGCA GGCTTGG GTCAGT OT3- TGCCAGTGAGTG 1673. 3 AGACACA 1674. GGTGGGCG 1675. 68C, 1 1 1 30 TGTGCGTGTGG GCCAGG TGTGTGTG 3% GCCTCAG TACC DMSO OT3- TGGGTGTGAGTG 1676. 3 ACACTCT 1677. GAGAAGTC 1678. 72C 1 2 0 31 TGTGCGTGTGG CACACA AGGGCTGG An- CGCACCA CGGG neal, A 3% DMSO OT3- TGTATGTGAGTG 1679. 3 ACTGCCT 1680. TGGTGAGG 1681. DMSO 1 1 1 32 TGTGCGTGTGG GCATTT GCTTCAGG CCCCGGT GAGC OT3- TGTGAGAGAGAG 1682. 3 GCCAGGT 1683. TCCTTCTA 1684. DMSO 2 1 0 33 TGTGCGTGTGG TCATTG CACATCGG ACTGCCC CGGC OT3- TGTGCCTGAGTG 1685. 3 CGAGGGA 1686. CTGACCTG 1687. DMSO 1 2 0 34 TGTGCGTGTGG GCCGAG GGGCTCTG TTCGTAA GTAC OT3- TGTGTGTGTGTG 1688. 3 TCCTCGG 1689. GCACTGAG 1690. DMSO 2 1 0 35 TGTGCGTGTGG GAAGTC CAACCAGG ATGGCTT AGCAC CA OT3- AGCGTGTGAGTG 1691. 4 Not  1 0 3 36 TATGCGTGGGG opti- mized OT3- ATTGAGTGTGTG 1692. 4 TAAACCG 1693. GCTCCCCT 1694. DMSO 2 1 1 37 AGTGCGTGGGG TTGCCC GCCAGGTG CCGCCTC AACC OT3- CATGTGTGGGTG 1695. 4 CCTGCTG 1696. CTGCGGAG 1697. DMSO 2 0 2 38 TGTGCGTGTGG AGACTC TGGCTGGC CAGGTCC TATA OT3- CCCGAGTGTGTG 1698. 4 CTCGGGG 1699. GGAGCAGC 1700. DMSO 3 0 1 39 TGTGCGTGTGG ACTGAC TCTTCCAG AAGCCGG GGCC OT3- CTGGAGTGAGTG 1701. 4 CCCCGAC 1702. CTGGCAGC 1703. DMSO 1 2 1 40 TGTGTGTGTGG CAAAGC CTCTGGAT AGGAGCA GGGG OT3- GTTTCATGAGTG 1704. 4 Not  0 3 1 41 TGTGCGTGGGG opti- mized OT3- TATGTGTGCGTG 1705. 4 ATTTCAG 1706. AGGCCGCG 1707. DMSO 1 2 1 42 TGTGCGTGTGG AGCCCC GTGTTATG GGGGAAA GTTA OT3- TATGTGTGTGTG 1708. 4 GCCAGTG 1709. TGACATAT 1710. DMSO 2 1 1 43 TGTGCGTGGGG GCTTAG TTTCCTGG TGTCTTT GCCATGGG GTGT T OT3- TCTGTGTGTGTG 1711. 4 TGCCAGA 1712. CCATGCTG 1713. DMSO 3 1 0 44 TGTGCGTGGGG AGAACA ACATCATA TGGGCCA TACTGGGA GA AGC OT3- TCTGTGTGTGTG 1714. 4 GCGTGTC 1715. CCAGGCTG 1716. DMSO 3 1 0 45 TGTGCGTGTGG TCTGTG GGCACACA TGCGTGC GGTT OT3- TGAGCGTGAGTG 1717. 4 Not  2 2 0 46 TGAGCGTGTGG opti- mized OT3- TGTCTTTGAGTG 1718. 4 TGCCCAG 1719. AGGATGAG 1720. DMSO 2 2 0 47 TGTGCGTGTGG TCCAAT TTCATGTC ATTTCAG CTTTGTGG CAGCT GG OT3- TTTGTGTGTGTG 1721. 4 GGGTGAA 1722 AATGACTC 1723. DMSO 2 2 0 48 TGTGCGTGTGG AATTTG ATTCCCTG GTACTGT GGTATCTC TAGCTG CCA T OT3- AAGGCGTGTGTG 1724. 5 TGCCCCA 1725. CAAGGTCG 1726. DMSO 1 2 2 49 TGTGCGTGTGG TCAATC GCAGGGCA ACCTCGG GTGA C OT3- AATTCGTGTGTG 1727. 5 GCCTCCT 1728. TGAGAGTT 1729. DMSO 1 2 2 50 TGTGCGTGGGG CTGCCG CCTGTTGC CTGGTAA TCCACACT OT3- ATGGTGTGTGTG 1730. 5 Not  2 2 1 51 TGTGCGTGTGG opti- mized OT3- CACGTGTGTGTG 1731. 5 GCCACCA  1732. ACATGCAT 1733. DMSO 3 0 2 52 TGTGCGTGTGG AAATAG CTGTGTGT CCAGCGT GCGT OT3- GAAATTTGAGTG 1734. 5 ACAGACT 1735. TGTATCTT 1736. DMSO 2 1 2 53 TGTGCGTGTGG GACCCT TCTTGCCA TGAAAAA ATGGTTTT TACCAG CCC T OT3- TAAGTGTGTGTG 1737. 5 AGCCAAA 1738. TCCTGGAG 1739. DMSO 3 1 1 54 TGTGCGTGTGG TTTCTC AGCAGGCA AACAGCA TTTTTGT GCACT OT3- TATATGTGTGTG 1740. 5 ACCTCCT 1741. GGCGGGAA 1742. DMSO 2 1 2 55 TGTGCGTGGGG TGTGCT GGTAACCC GCCTGGC TGGG OT3- TATCTGTGTGTG 1743. 5 CACAAAG 1744. TGATCCGA 1745. DMSO 3 1 1 56 TGTGCGTGTGG CTCTAC TGGTTGTT CTTTCCA CACAGCT GTAGTG T OT3- TTTATGTGTGTG 1746. 5 TGTGGGG 1747. ACGCACAA 1748. DMSO 2 2 1 57 TGTGCGTGTGG ATTACC AAATGCCC TGCCTGG TTGTCA C OT3- TTTTTGTGTGTG 1749. 5 TGAGGCA 1750. GCCCGAGC 1751. DMSO 2 3 0 58 TGTGCGTGGGG GACCAG ACAGTGTA TCATCCA GGGC GC OT3- AAAAATTGTGTG 1752. 6 ATTAGCT 1753. ACTGCATC 1754. DMSO 2 1 3 59 TGTGCGTGGGG GGGCGT TCATCTCA GGCGGAG GGCAGCT OT3- ACAATGTGTGTG 1755. 6 TGAAGCA 1756. TCAGCTTC 1757. DMSO 4 0 2 60 TGTGCGTGTGG GAAGGA ACATCTGT GTGGAGA TTCAGTTC AGGA AGT OT3- ATGTGGTGTGTG 1758. 6 TGGTGGA 1759. AGAGCAGA 1760. DMSO 1 3 2 61 TGTGCGTGTGG GTGTGT AAGAGAGT GTGTGGT GCCCA OT3- CAAAATTGTGTG 1761. 6 GCCCCTG 1762. TGCACAAG 1763. DMSO 3 1 2 62 TGTGCGTGTGG TACGTC CCACTTAG CTGACAG CCTCTCT C OT3- CCCTGGTGTGTG 1764. 6 AGCGCAG 1765. TCTCTCGC 1766. DMSO 3 1 2 63 TGTGCGTGTGG GTAAAC CCCGTTTC AGGCCCA CTTGT OT3- TCCGCTTGTGTG 1767. 6 ATGGGTG 1768. ACAGCAGG 1769. DMSO 2 3 1 64 TGTGCGTGGGG CCAGGT AAGGAGCC ACCACGC GCAG OT3- TCCTCGTGTGTG 1770. 6 CGGGCGG 1771. AGGAGGTC 1772. DMSO 2 3 1 65 TGTGCGTGTGG GTGGAC TCGAGCCA AGATGAG GGGG OT3- TTAAGGTGGGTG 1773. 6 TCAACCT 1774. GTCTATAT 1775. DMSO 1 2 3 66 TGTGCGTGGGG AGTGAA ACAGCCCA CACAGAC CAACCTCA CACTGA TGT OT3- TTATATTGTGTG 1776. 6 GCCAGGG 1777. TGTCATTT 1778. DMSO 2 4 0 67 TGTGCGTGGGG CCAGTG CTTAGTAT GATTGCT GTCAGCCG GA OT3- TTGAGGAGAGTG 1779. 6 GAGCCCC 1780. GCCAGAGC 1781. DMSO 1 3 2 68 TGTGCGTGAGG ACCGGT TACCCACT TCAGTCC CGCC 1782. 1783. 1784. Tar-  GAGTCCGAGCAG 1785. 0 GGAGCAG 1786. GGGAAGGG 1787. DMSO get AAGAAGAAGGG CTGGTC GGACACTG 4 AGAGGGG GGGA OT4- GAGTTAGAGCAG 1788. 2 TCTCTCC 1789. ATCTGCAC 1790. DMSO 0 1 1 1 AAGAAGAAAGG TTCAAC ATGTATGT TCATGAC ACAGGAGT CAGCT CAT OT4- AAGTCAGAGGAG 1791. 3 AAGACAG TGGGGAA 1792. AGGGTGTA 1793. DMSO 2 1 1 2 AAGAAGAAGGG AGGAGAA TCTCCA CTGTGGGA GAAGAAG   AAGAACC ACTTTGCA GG CCC (SEQ  ID NO:  2229) OT4- AAGTCCGAGGAG 1794. 3 GATGGCC 1795. ACTTCGTA 1796. DMSO 1 0 2 3 AGGAAGAAAGG CCACTG GAGCCTTA AGCACGT AACATGTG GC OT4- AAGTCTGAGCAC 1797. 3 AGGATTA 1798. TCAAACAA 1799. 1M 1 0 2 4 AAGAAGAATGG ATGTTT GGTGCAGA be- AAAGTCA TACAGCA taine, CTGGTG TD G OT4- ACGTCTGAGCAG 1800. 3 TCCAAGC 1801. TGCTCTGT 1802. DMSO 0 1 2 5 AAGAAGAATGG CACTGG GGATCATA TTTCTCA TTTTGGGG GTCA GA OT4- GACTCCTAGCAA 1803. 3 ACTTTCA 1804. CCCACGCT 1805. DMSO 1 1 1 6 AAGAAGAATGG GAGCTT GAAGTGCA GGGGCAG ATGGC GT OT4- GAGACTGAGAAG 1806. 3 CAAAGCA 1807. GGCTCTTC 1808. 1M 1 1 1 7 AAGAAGAAAGG TGCCTT GATTTGGC be- TCAGCCG ACCT taine, TD OT4- GAGCCGGAGCAG 1809. 3 Not  1 0 2 8 AAGAAGGAGGG opti- mized OT4- GAGCCTGAGCAG 1810. 3 GGACTCC 1811. AGGAACAC 1812. 72C 0 0 3 9 AAGGAGAAGGG CTGCAG AGGCCAGG An- CTCCAGC CTGG neal, 6% DMSO OT4- GAGGCCGAGCAG 1813. 3 CCCTTTA 1814. CCGACCTT 1815. DMSO 0 1 2 10 AAGAAAGACGG GGCACC CATCCCTC TTCCCCA CTGG OT4- GAGTAAGAGAAG 1816. 3 TGATTCT 1817. TGGGCTCT 1818. DMSO 0 3 0 11 AAGAAGAAGGG GCCTTA GTGTCCCT GAGTCCC ACCCA AGGT OT4- GAGTAGGAGGAG 1819. 3 Not  2 1 0 12 AAGAAGAAAGG opti- mized OT4- GAGTCCGGGAAG 1820. 3 AGGCAGG 1821. ACCCTGAC 1822. DMSO 0 1 2 13 GAGAAGAAAGG AGAGCA TACTGACT AGCAGGT GACCGCT OT4- GATTCCTACCAG 1823. 3 CTCCCCA 1824. AGAGGCAT 1825. DMSO 1 2 0 14 AAGAAGAATGG TTGCGA TGACTTGG CCCGAGG AGCACCT OT4- GCGACAGAGCAG 1826. 3 CTGGAGC 1827. CCTCAGGG 1828. DMSO 1 2 0 15 AAGAAGAAGGG CCAGCA AGGGGGCC GGAAGGC TGAT OT4- AAATCCAACCAG 1829. 4 ACTGTGG 1830. AGGTCGGT 1831. DMSO 1 0 3 16 AAGAAGAAAGG GCGTTG GCAGGGTT TCCCCAC TAAGGA OT4- AAGTCTGAGGAC 1832. 4 GGCGCTC 1833. CGTCACCC 1834. DMSO 2 0 2 17 AAGAAGAATGG CCTTTT ATCGTCTC TCCCTTT GTGGA GT OT4- AAGTTGGAGCAG 1835. 4 TGCCATC 1836. GCATCTTG 1837. DMSO 1 0 3 18 GAGAAGAAGGG TATAGC CTAACCGT AGCCCCC ACTTCTTC T TGA OT4- AATACAGAGCAG 1838. 4 GTGGAGA 1839. GCTCCTGG 1840. DMSO 1 2 1 19 AAGAAGAATGG CGCTAA CCTCTTCC ACCTGTG TACAGC AGGT OT4- AGGTACTAGCAG 1841. 4 CCGAACT 1842. CCAAGTCA 1843. DMSO 0 2 2 20 AAGAAGAAAGG TCTGCT ATGGGCAA GAGCTTG CAAGGGA ATGC OT4- AGGTGCTAGCAG 1844. 4 Not  1 1 2 21 AAGAAGAAGGG opti- mized OT4- AGGTGGGAGCAG 1845. 4 TGCCCCC 1846. ATGGCAGG 1847. DMSO 2 0 2 22 AAGAAGAAGGG AAGACC CAGAGGAG TTTCTCC GAAG OT4- CAAACGGAGCAG 1848. 4 GGGTGGG 1849. CTGGGGCC 1850. DMSO 3 0 1 23 AAGAAGAAAGG GCCATT AGGGTTTC GTGGGTT TGCC OT4- CACTCTGAGGAG 1851. 4 TGGAGAA 1852. TCCTTCTG 1853. DMSO 3 0 1 24 AAGAAGAAAGG CATGAG TAGGCAAT AGGCTTG GGGAACAA CAA OT4- CAGTCATGGCAG 1854. 4 GCCACAT 1855. GGCAGATT 1856. 1M 1 2 1 25 AAGAAGAAAGG GGTAGA TCCCCCAT be- AGTCGGC GCTG taine, TD OT4- CCGTCCCAGCAG 1857. 4 TGTACAC 1858. AAGGGGAG 1859. DMSO 3 1 0 26 TAGAAGAATGG CCCAAG TGTGCAAG TCCTCCC CCTC OT4- GTCTGCGATCAG 1860. 4 AGGTCTG 1861. AGTCCAAC 1862. DMSO 3 1 0 27 AAGAAGAAAGG GCTAGA ACTCAGGT GATGCAG GAGACCCT CA OT4-  TAATCCAATCAG 1863. 4 CCAAGAG 1864. GGGTATGG 1865. DMSO 0 2 2 28 AAGAAGAAGGG GACCCA AATTCTGG GCTGTTG ATTAGCAG GA AGC OT4- TATACGGAGCAG 1866. 4 ACCATCT 1867. ACACTGTG 1868. DMSO 2 2 0 29 AAGAAGAATGG CTTCAT AGTATGCT TGATGAG TGGCGT TCCCAA OT4- ACTTCCCTGCAG 1869. 5 GGCTGCG 1870. TCGGATGC 1871. DMSO 2 2 1 30 AAGAAGAAAGG GGGAGA TTTTCCAC TGAGCTC AGGGCT OT4- AGGACTGGGCAG 1872. 5 TCTTCCA 1873. CCAATCCT 1874. DMSO 1 0 4 31 AAGAAGAAGGG GGAGGG GAGCTCCT CAGCTCC ACAAGGCT OT4- AGGTTGGAGAAG 1875. 5 GAGCTGC 1876. TGCTGGTT 1877. DMSO 1 1 3 32 AAGAAGAAGGG ACTGGA AAGGGGTG TGGCACT TTTTGGA OT4- AGTTCAGAGCAG 1878. 5 TCTGGGA 1879. TGGGGGAC 1880. DMSO 0 2 3 33 GAGAAGAATGG AGGTGA AATGGAAA GGAGGCC AGCAATGA A OT4- ATGACACAGCAG 1881. 5 CTTGCTC 1882. AGCCCTTG 1883. DMSO 3 1 1 34 AAGAAGAAGGG CCAGCC CCATGCAG TGACCCC GACC OT4- ATGACAGAGAAG 1884. 5 GGGATTT 1885. AACCACAG 1886. DMSO 2 2 1 35 AAGAAGAAAGG TTATCT ATGTACCC GTTGGGT TCAAAGCT GCGAA OT4- CCGCCCCTGCAG 1887. 5 ACCCATC 1888. TCTGGAAC 1889. 72C 3 1 1 36 AAGAAGAACGG AGGACC CTGGGAGG An- GCAGCAC CGGA neal, 3% DMSO OT4- GCAGGAGAGCAG 1890. 5 CGTCCCT 1891. CCTCCTTG 1892. DMSO 1 3 1 37 AAGAAGAAAGG CACAGC GGCCTGGG CAGCCTC GTTC OT4- GTTCAAGAGCAG 1893. 5 CCCTCTG 1894. AGATGTTC 1895. DMSO 1 3 1 38 AAGAAGAATGG CAAGGT TGTCCCCA GGAGTCT GGCCT CC OT4- GTTTTGAAGCAG 1896. 5 GGCTTCC 1897. TGCCGCTC 1898. DMSO 2 1 2 39 AAGAAGAAAGG ACTGCT CACATACC GAAGGCC CTCC T OT4- TATGGCAAGCAG 1899. 5 AGCATTG 1900. AGCACCTA 1901. DMSO 1 3 1 40 AAGAAGAAAGG CCTGTC TTGGACAC GGGTGAT TGGTTCTC GT T OT4- TGGTGGGATCAG 1902. 5 TCTAGAG 1903. TGGAGATG 1904. DMSO 2 2 1 41 AAGAAGAAAGG CAGGGG GAGCCTGG CACAATG TGGGA C OT4- ACCCACGGGCAG 1905. 6 GGTCTCA 1906. CCCACAGA 1907. DMSO 1 2 3 42 AAGAAGAAGGG GAAAAT AACCTGGG GGAGAGA CCCT AAGCAC G OT4- ACTCCTGATCAG 1908. 6 GGTTGCT 1909. TGGGTCCT 1910. DMSO 0 3 3 43 AAGAAGAAGGG GATACC CTCCACCT AAAACGT CTGCA TTGCCT OT4- ACTGATGAGCAG 1911. 6 ACTCTCC 1912. CAGAATCT 1913. DMSO 0 4 2 44 AAGAAGAAAGG TTAAGT TGCTCTGT ACTGATA TGCCCA TGGCTG T OT4- ATTTTAGTGCAG 1914. 6 Not  2 2 2 45 AAGAAGAAAGG opti- mized OT4- ATTTTAGTGCAG 1915. 6 Not  2 2 2 46 AAGAAGAAAGG opti- mized OT4- CCATGGCAGCAG 1916. 6 CAATGCC 1917. TCCCAAGA 1918. DMSO 4 1 1 47 AAGAAGAAGGG TGCAGT GAAAACTC CCTCAGG TGTCCTGA A CA OT4- CCATTACAGCAG 1919. 6 GCATTGG 1920. TGGCTGTG 1921. DMSO 2 2 2 48 AAGAAGAAGGG CTGCCC CTGGGCTG AGGGAAA TGTT OT4- CGAGGCGGGCAG 1922. 6 CCACAAG 1923. ACAGGTGC 1924. DMSO 2 1 3 49 AAGAAGAAAGG CCTCAG CAAAACAC CCTACCC TGCCT G OT4- TCATTGCAGCAG 1925. 6 TCATTGC GCCTCTT 1926. CGATCAGT 1927. DMSO 2/ 2/3 2 50 AAGAAGAAAGG AGCAGAA GCAAAT CCCCTGGC 1 GAAGAAA GAGACTC GTCC GG CTTTT TCATTGT  AGCAGAA GAAGAAA  GG (SEQ  ID NO:  2230) OT4- TCTCCAGGGCAG 1928. 6 TCCCAGA 1929. AGGGGTTT 1930. DMSO 0 4 2 51 AAGAAGAAAGG ATCTGC CCAGGCAC CTCCGCA ATGGG 1931. 1932. 1933. Tar-  GTCATCTTAGTC 1934. 0 TCCTAAA 1935 AAAGTGTT 1936. DMSO get ATTACCTGAGG AATCAG AGCCAACA 5 TTTTGAG TACAGAAG ATTTAC TCAGGA TTCC OT5- GGTATCTAAGTC 1937. 3 GGTATCT ACATCTG 1938. TGTCTGAG 1939. DMSO 1/ 1 1 1 ATTACCTGTGG AAGTCAT GGGAAA TATCTAGG 2 TACCTGT GCAAAAG CTAAAAGT GG TCAACA GGT (SEQ   ID NO:   2231) GGTATCT  AAGTCAA TACCTGT GG (SEQ  ID NO:  2232) OT5- GTAATATTAGTC 1940. 3 ACGATCT 1941. AGTGCTTT 1942. DMSO 0 3 0 2 ATTACCGGTGG TGCTTC GTGAACTG ATTTCCC AAAAGCAA TGTACA ACA OT5- GTAATCTGAGTC 1943. 3 GCACCTT 1944. GGGCAACT 1945. DMSO 1 2 0 3 ATTTCCTGGGG GGTGCT GAACAGGC GCTAAAT ATGAATGG GCC OT5- GTCATCCTAGTC 1946. 3 AACTGTC 1947. GGTGCACC 1948. DMSO 1 1 1 4 ATTTACTGGGG CTGCAT TGGATCCA CCCCGCC CCCA OT5- GTCATCCTAGTG 1949. 3 Not  1950. 1951. 1 1 1 5 CTTACCTGAGG opti- mized OT5- GTCATCTGAGGC 1952. 3 CATCACC 1953. ACCACTGC 1954. 72C 0 3 0 6 ATTAACTGAGG CTCCAC TGCAGGCT An- CAGGCCC CCAG neal, 3% DMSO OT5- AATATGTTAGTC 1955. 4 Not  2 0 2 7 ATTACCTGAGG opti- mized OT5- ATAAACGTAGTC 1956. 4 CCTGACC 1957. TGGTGCGT 1958. 72C 1 2 1 8 ATTACCTGAGG CGTGGT GGTGTGTG An- TCCCGAC TGGT neal, 3% DMSO OT5- ATCATCATCGTC 1959. 4 TGGGAAC 1960. CCATGTGA 1961. DMSO 1 1 2 9 ATTATCTGGGG ATTGGA CTACTGGG GAAGTTT CTGCCC CCTGA OT5- ATCATTTTACTC 1962. 4 AGCCTTG 1963. GGTTCTCT 1964. DMSO 1 0 3 10 ATTACTTGTGG GCAAGC CTCTCAGA AACTCCC AAAGAAAG T AGG OT5- ATCATTTTAGTC 1965. 4 GGCAGCG 1966. GCCAGAGG 1967. DMSO 1 0 3 11 ATCTCCTGTGG GACTTC CTCTCAGC AGAGCCA AGTGC OT5- CACAGCTTAGTC 1968. 4 CCAGCCT 1969. ACTGTGCC 1970. DMSO 2 1 1 12 ATCACCTGGGG GGTCAA CAGCCCCA TATGGCA TATT OT5- CCCAGCTTAGTC 1971. 4 ATGCCAA 1972. CGGGTTGT 1973. DMSO 2 1 1 13 ATTAGCTGTGG CACTCG GGCACCGG AGGGGCC GTTA OT5- CTCACCTTTGTC 1974. 4 TTGCTCT 1975. AGAGTTCA 1976. DMSO 3 0 1 14 ATTTCCTGAGG AGTGGG GGCATGAA GAGGGGG AAGAAGCA ACA OT5- CTCATTTTATTC 1977. 4 AGCTGAA 1978. TGCAATTT 1979. DMSO 1 1 2 15 ATTGCCTGGGG GATAGC GAGGGGCT AGTGTTT CTCTTCA AAGCCT OT5- CTCTCCTTAGTC 1980. 4 AGTCACT 1981. TGCCAGCC 1982. DMSO 2 0 2 16 ACTACCTGAGG GGAGTA AAAAGTTG AGCCTGC TTAGTGTG CT T OT5- CTTATCTCTGTC 1983. 4 GGGTCTC 1984. TGTGTGGT 1985. DMSO 2 0 2 17 ATTACCTGGGG CCTCAG AGGGAGCA TGCCCTG AAACGACA OT5- GACAGCTCCGTC 1986. 4 TGGGGGC 1987. TGACCACA 1988. DMSO 1 2 1 18 ATTACCTGGGG TGTTAA CACACCCC GAGGCAC CACG A OT5- GCCACCTCAGTC 1989. 4 TCAAAAC 1990. TGTGTTTT 1991. DMSO 1 0 3 19 ATTAGCTGGGG AGATTG TAAGCTGC ACCAAGG ACCCCAGG CCAAAT OT5- GGAATCTTACTC 1992. 4 TCTGGCA 1993. GCACGCAG 1994. DMSO 1 2 1 20 ATTACTTGGGG CCAGGA CTGACTCC CTGATTG CAGA TACA OT5- GTGGCCTCAGTC 1995. 4 Not  1 0 3 21 ATTACCTGCGG opti- mized OT5- GTTGTTTTAGTG 1996. 4 AGCATCT 1997. ACCAGGGC 1998. DMSO 1 0 3 22 ATTACCTGAGG GTGATA TGCCACAG CCCTACC AGTC TGTCT OT5- TACATCTTAGTC 1999. 4 TAGTCTT 2000. CTCGGCCC 2001. DMSO 1 2 1 23 CTCACCTGTGG GTTGCC CTGAGAGT CAGGCTG TCAT OT5- TCCATCTCACTC 2002. 4 TCCATCT CTGCAAC 2003. GAGCAGCA 2004. DMSO 1 1 2 24 ATTACCTGAGG CACTCAT  CAGGGC GCAAAGCC TACCTGA CCTTACC ACCG GG (SEQ   ID NO:  2233) TCCATCT CACTCAT TACCTGA  TG (SEQ  ID NO:  2234) OT5- TTCATCCTAGTC 2005. 4 GCCTGGA 2006. AGCCGAGA 2007. DMSO 1 1 2 25 AACACCTGGGG GAGCAA CAATCTGC GCCTGGG CCCG OT5- TTTATATTAGTG 2008. 4 TTTATAT AGTGAAA 2009. GGCAGGTC 2010. No 1 2 1 26 ATTACCTGTGG TAGTGAT  CAAACA TGACCAGT DMSO TACCTGC  AGCAGCA GGGG TD GG GTCTGA (SEQ  ID NO:  2235) OT5- AACGTGTAAGTC 2011. 5 AGGCTCA 2012. TGAGTAGA 2013. DMSO 3 0 2 27 ATTACCTGAGG GAGAGG CAGAAATG TAAGCAA TTACCGGT TGGA GTT OT5- AAGATCACAGTC 2014. 5 TCAGAGA 2015. AGTGAACC 2016. DMSO 3 0 2 28 ATTACCTGGGG TGTTAA AAGGGAAT AGCCTTG GGGGGA GTGGG OT5- AGAATATTAGTC 2017. 5 TGTGCTT 2018. CACCTCAG 2019. DMSO 0 4 1 29 CTTACCTGGGG CTTGGG CCCTGTAG GTAGTGG TCCTGG CA OT5- AGCAGATTAGTG 2020. 5 CCATTGG 2021. GCCACTGT 2022. 1M 1 3 1 30 ATTACCTGGGG GTGACT CCCCAGCC be- GAATGCA TATT taine, CA TD OT5- AGTAGCTTAGTG 2023. 5 ACCAAGA 2024. TGAGATGG 2025. DMSO 1 2 2 31 ATTACCTGGGG AAGTGA CATACGAT AAAGGAA TTACCCA ACCC OT5- CACGGCTTACTC 2026. 5 AGGGTGG 2027. TGGCATCA 2028. DMSO 3 1 1 32 ATTACCTGGGG GGACTG CTCAGAGA AAAGGAG TTGGAACA CT CA OT5- CATATGTTAGGC 2029. 5 ACCAGTG 2030. TCCTATGG 2031. DMSO 3 1 1 33 ATTACCTGGGG CTGTGT GAGGGGAG GACCTTG GCTTCT GA OT5- CATTTCTTAGTC 2032. 5 CCAGGTG 2033. GCATACGG 2034. 68C, 4 0 1 34 ATTTCCTGAGG TGGTGG CAGTAGAA 3% TTCATGA TGAGCC DMSO C OT5- TGCAGCTAACTC 2035. 5 CAGGCGC 2036. CCTTCCTG 2037. DMSO 2 3 0 35 ATTACCTGCGG TGGGTT GGCCCCAT CTTAGCC GGTG T OT5- TTGCTTTTAGTT 2038. 5 TGGGGTC 2039. TGAAACTG 2040. DMSO 1 2 2 36 ATTACCTGGGG CAAGAT CTTGATGA GTCCCCT GGTGTGGA OT5- AACTTGAAAGTC 2041. 6 GCTGGGC 2042. ACTTGCAA 2043. DMSO 5 0 1 37 ATTACCTGTGG TTGGTG AGCTGATA GTATATG ACTGACTG C A OT5- AAGGTCACAGTC 2044. 6 AGTTGGT 2045. CGCAGCGC 2046. DMSO 3 0 3 38 ATTACCTGGGG GTCACT ACGAGTTC GACAATG ATCA GGA OT5- AATGTCTTCATC 2047. 6 AGAGGAG 2048. GGCTGGGG 2049. DMSO 1 1 4 39 ATTACCTGAGG GCACAA AGGCCTCA TTCAACC CAAT CCT OT5- AGATGCTTGGTC 2050. 6 GGGAAAG 2051. AGGACAAG 2052. DMSO 1 3 2 40 ATTACCTGTGG TTTGGG CTACCCCA AAAGTCA CACC GCA OT5- AGTAGATTAGTT 2053. 6 TGGTGCA 2054. TCATTCCA 2055. DMSO 0 3 3 41 ATTACCTGGGG TCAAAG GCACGCCG GGTTGCT GGAG TCT OT5- AGTAGGTTAGTA 2056. 6 CCCAGGC 2057. TGGAGTAA 2058. DMSO 1 3 2 42 ATTACCTGGGG TGCCCA GTATACCT TCACACT TGGGGACC T OT5- CAAATGAGAGTC 2059. 6 TCAGTGC 2060. TGTGCAAA 2061. DMSO 4 2 0 43 ATTACCTGAGG CCCTGG TACCTAGC GTCCTCA ACGGTGC OT5- CATGTCTGAATC 2062. 6 AGCACTC 2063. ACTGAAGT 2064. DMSO 2 1 3 44 ATTACCTGAGG CCTTTT CCAGCCTC GAATTTT TTCCATTT GGTGCT CA OT5- CCTGACTTGGTC 2065. 6 GAAACCG 2066. GGGGAGTA 2067. DMSO 2 0 4 45 ATTACCTGTGG GTCCCT GAGGGTAG GGTGCCA TGTTGCC OT5- CGTGCATTAGTC 2068. 6 TTGCGGG 2069. AGGTGCCG 2070. DMSO 1 2 3 46 ATTACCTGAGG TCCCTG TGTTGTGC TGGAGTC CCAA Tar-  GGAATCCCTTCT 2071. 0 GCCCTAC 2072. GGGCCGGG 2073. DMSO get GCAGCACCTGG ATCTGC AAAGAGTT 6 TCTCCCT GCTG CCA OT6- GGAACCCCGTCT 2074. 2 TTGGAGT 2075. ACCTCTCT 2076. DMSO 0 1 1 1 GCAGCACCAGG GTGGCC TTCTCTGC CGGGTTG CTCACTGT OT6- GGAACACCTTCT 2077. 3 CACACCA 2078. GCAGTACG 2079. DMSO 1 1 1 2 GCAGCTCCAGG TGCTGA GAAGCACG TCCAGGC AAGC OT6- GGAAGCTCTGCT 2080. 3 CTCCAGG 2081. CTGGGCTC 2082. DMSO 0 2 1 3 GCAGCACCTGG GCTCGC TGCTGGTT TGTCCAC CCCC OT6- GGAATATCTTCT 2083. 3 CTGTGGT 2084. CCCCATAC 2085. DMSO 0 2 1 4 GCAGCCCCAGG AGCCGT CACCTCTC GGCCAGG CGGGA OT6- GGAATCACTTTT 2086. 3 GGTGGCG 2087. CCAGCGTG 2088. 1M 0 1 2 5 ACAGCACCAGG GGACTT TTTCCAAG be- GAATGAG GGAT taine, TD OT6- GGAATCCCCTCT 2089. 3 GGAATCC CCAGAGG 2090. TTTCCACA 2091. DMSO 1 1 1/2 6 CCAGCCCCTGG CCTCTCC  TGGGGC CTCAGTTC AGCCCCT  CCTGTGA TGCAGGA GG (SEQ  ID NO:  2236) GGAATCC CCTCTCC AGCCTCT GG (SEQ   ID NO:  2237) OT6- GGAATCTCTTCT 2092. 3 GGAATCT TGTGACT 2093. GCAGTGTT 2094. 1M 0 1 5 7 TCAGCATCTGG CTTCCTT GGTTGT TTGTGGTG be- GGCATCT  CCTGCTT ATGGGCA taine, GG TCCT TD (SEQ  ID NO:  2238) OT6- GGAATTGCTTCT 2095. 3 CTGGCCA 2096. TGGGACCC 2097. DMSO 1 0 2 8 GCAGCGCCAGG AGGGGT CAGCAGCC GAGTGGG AATG OT6- GGACTCCCCTCT 2098. 3 ACGGTGT 2099. ACAGTGCT 2100. DMSO 1 1 1 9 GCAGCAGCTGG GCTGGC GACCGTGC TGCTCTT TGGG OT6- GGAGTCCCTCCT 2101. 3 TGGTTTG 2102. TGCCTCCC 2103. DMSO 0 0 3 10 ACAGCACCAGG GGCCTC ACAAAAAT AGGGATG GTCTACCT G OT6- GGAGTCCCTCCT 2104. 3 TGGTTTG 2105. ACCCCTTA 2106. DMSO 0 0 3 11 ACAGCACCAGG GGCCTC TCCCAGAA AGGGATG CCCATGA G OT6- GGCATCCATTCT 2107. 3 TCCAAGT 2108. TGGGAGCT 2109. DMSO 0 3 0 12 GCAGCCCCTGG CAGCGA GTTCCTTT TGAGGGC TTGGCCA T OT6- GGCTTCCCTTCT 2110. 3 CACCCCT 2111. GCTAGAGG 2112. DMSO 1 2 0 13 GCAGCCCCAGG CTCAGC GTCTGCTG TTCCCAA CCTT OT6- TGAATCCCATCT 2113. 3 AGACCCC 2114. CTTGCTCT 2115. DMSO 2 1 0 14 CCAGCACCAGG TTGGCC CACCCCGC AAGCACA CTCC OT6- AAAATACCTTCT 2116. 4 ACATGTG 2117. TCTCACTT 2118. DMSO 0 1 3 15 GCAGTACCAGG GGAGGC TGCTGTTA GGACAGA CCGATGTC G OT6- AAAATCCCTTCT 2119. 4 GGACGAC 2120. AGTGCCCA 2121. 72C 0 1 3 16 TCAACACCTGG TGTGCC GAGTGTTG An- TGGGACA TAACTGCT neal, 3% DMSO OT6- ACACTCCCTCCT 2122. 4 GGAGAGC 2123. CAGCGTGG 2124. DMSO 1 1 2 17 GCAGCACCTGG TCAGCG CCCGTGGG CCAGGTC AATA OT6- ACCATCCCTCCT 2125. 4 GCTGAAG 2126. ACCCCACT 2127. DMSO 1 1 2 18 GCAGCACCAGG TGCTCT GTGGATGA GGGGTGC ATTGGTAC T C OT6- AGAGGCCCCTCT 2128. 4 TCGGGGT 2129. TTGCCTCG 2130. DMSO 0 1 3 19 GCAGCACCAGG GCACAT CAGGGGAA GGCCATC GCAG OT6- AGGATCCCTTGT 2131. 4 CTCGTGG 2132. AGCCACCA 2133. DMSO 2 0 2 20 GCAGCTCCTGG GAGGCC ACACATAC AACACCT CAGGCT OT6- CCACTCCTTTCT 2134. 4 GCATGCC 2135. AGGATTTC 2136. DMSO 2 1 1 21 GCAGCACCAGG TTTAAT AGAGTGAT CCCGGCT GGGGCT OT6- GAAGGCCCTTCA 2137. 4 CGCCCAG 2138. GCAAATTT 2139. DMSO 1 1 2 22 GCAGCACCTGG CCACAA CTGCACCT AGTGCAT ACTCTAGG CCT OT6- GATATCCCTTCT 2140. 4 AGCTCAC 2141. GCAGTCAC 2142. DMSO 1 1 2 23 GTATCACCTGG AAGAAT CCTTCACT TGGAGGT GCCTGT AACAGT OT6- GGGTCCGCTTCT 2143. 4 AAACTGG 2144. GGGGCTAA 2145. DMSO 2 0 2 24 GCAGCACCTGG GCTGGG GGCATTGT CTTCCGG CAGACCC OT6- GTCTCCCCTTCT 2146. 4 GCAGGTA 2147. TCTCCTGC 2148. 1M 1 2 1 25 GCAGCACCAGG GGCAGT CTCAGCCT be- CTGGGGC CCCA taine, TD OT6- GTCTCCCCTTCT 2149. 4 GCAGGTA 2150. TCTCCTGC 2151. 1M 1 2 1 26 GCAGCACCAGG GGCAGT CTCAGCCT be- CTGGGGC CCCA taine, TD OT6- GTCTCCCCTTCT 2152. 4 GCAGGTA 2153. TCTCCTGC 2154. 1M 1 2 1 27 GCAGCACCAGG GGCAGT CTCAGCCT be- CTGGGGC CCCA taine, TD OT6- TCATTCCCGTCT 2155. 4 GCTCTGG 2156. GGCCTGTC 2157. DMSO 2 2 0 28 GCAGCACCCGG GGTAGA AACCAACC AGGAGGC AACC OT6- TGCACCCCTCCT 2158. 4 TGACATG 2159. AAATCCTG 2160. DMSO 0 2 2 29 GCAGCACCAGG TTGTGT CAGCCTCC GCTGGGC CCTT OT6- TGCATACCCTCT 2161. 4 TCCTGGT 2162. TCCTCCCC 2163. DMSO 0 3 1 30 GCAGCACCAGG GAGATC ACTCAGCC GTCCACA TCCC GGA OT6- TGCATGGCTTCT 2164. 4 TCCTAAT 2165. AGGGACCA 2166. DMSO 2 2 0 31 GCAGCACCAGG CCAAGT GCCACTAC CCTTTGT CCTTCA TCAGAC A OT6- AATATTCCCTCT 2167. 5 GGGACAC 2168. GGGGGAGA 2169. DMSO 1 0 4 32 GCAGCACCAGG CAGTTC TTGGAGTT CTTCCAT CCCC OT6- ACCATTTCTTCT 2170. 5 ACACCAC 2171. TCTGCCTG 2172. DMSO 1 1 3 33 GCAGCACCTGG TATCAA GGGTGCTT GGCAGAG TCCC TAGGT OT6- AGCTCCCATTCT 2173. 5 CTGGGAG 2174. GCCCCGAC 2175. DMSO 1 2 2 34 GCAGCACCCGG CGGAGG AGATGAGG GAAGTGC CCTC OT6- CAGATTCCTGCT 2176. 5 CAGATTA CGGGTCT 2177. ACCCAGGA 2178. DMSO 1 2 3 35 GCAGCACCGGG CTGCTGC CGGAAT ATTGCCAC AGCACCG  GCCTCCA CCCC GG (SEQ   ID NO:  2239) OT6- CCAAGAGCTTCT 2179. 5 TTGCTGT 2180. GCAGACAC 2181. DMSO 3 2 0 36 GCAGCACCTGG GGTCCC TAGAGCCC GGTGGTG GCCC OT6- CCCAGCCCTGCT 2182. 5 GGTGTGG 2183. ACCTGCGT 2184. DMSO 2 3 0 37 GCAGCACCCGG TGACAG CTCTGTGC GTCGGGT TGCA OT6- CCCCTCCCTCCT 2185. 5 CTCCCAG 2186. CCTGGCCC 2187. DMSO 2 2 1 38 GCAGCACCGGG GACAGT CATGCTGC GCTGGGC CTG OT6- CTACTGACTTCT 2188. 5 TGCGTAG 2189. AGGGAATG 2190. DMSO 2 3 0 39 GCAGCACCTGG GTTTTG ATGTTTTC CCTCTGT CACCCCCT GA OT6- CTCCTCCCTCCT 2191. 5 CTCCGCA 2192. TGCATTGA 2193. DMSO 1 3 1 40 GCAGCACCTGG GCCACC CGTACGAT GTTGGTA GGCTCA OT6- TCTGTCCCTCCT 2194. 5 ACCTGCA 2195. ACCTGAGC 2196. DMSO 2 1 2 41 GCAGCACCTGG GCATGA AACATGAC ACTCTCG TCACCTGG CA OT6- ACACAAACTTCT 2197. 6 ACACAAA TCTCCAG 2198. ACCATTGG 2199. 1M 3/ 3 1 42 GCAGCACCTGG CTTCTGC TTTCTT TGAACCCA be- 2 AGCACCT GCTCTCA GTCA taine, GG TGG TD ACACAAA CTTCTGC AGCACGT GG (SEQ  ID NO:  2240) OT6- ACTGTCATTTCT 2200. 6 TGGGGTG 2201. TCAGCTAT 2202. DMSO 2 1 3 43 GCAGCACCTGG GTGGTC AACCTGGG TTGAATC ACTTGTGC CA T OT6- ACTTTATCTTCT 2203. 6 AGCAGCC 2204. CCCTTTCA 2205. DMSO 3 1 2 44 GCAGCACCTGG AGTCCA TCGAGAAC GTGTCCT CCCAGGG G OT6- ATCCTTTCTTCT 2206. 6 TGGACGC 2207. GAGGTCTC 2208. DMSO 0 3 3 45 GCAGCACCTGG TGCTGG GGGCTGCT GAGGAGA CGTG OT6- CACCACCGTTCT 2209. 6 AGGTTTG 2210. TGGGGTGA 2211. DMSO 3 2 1 46 GCAGCACCAGG CACTCT TTGGTTGC GTTGCCT CAGGT GG OT6- CATGTGGCTTCT 2212. 6 TCTTCCT 2213. TGCAGGAA 2214. DMSO 4 0 2 47 GCAGCACCTGG TTGCCA TAGCAGGT GGCAGCA ATGAGGAG CA T OT6- CATTTTCTTTCT 2215. 6 GGACGCC 2216. GCCCTGGC 2217. DMSO 3 0 3 48 GCAGCACCTGG TACTGC AGCCCATG CTGGACC GTAC OT6- CTCTGTCCTTCT 2218. 6 AGGCAGT 2219. GGTCCCAC 2220. DMSO 2 3 1 49 GCAGCACCTGG CATCGC CTTCCCCT CTTGCTA ACAA OT6- CTGTACCCTCCT 2221. 6 Not  3 1 2 50 GCAGCACCAGG opti- mized OT6- TTGAGGCCGTCT 2222. 6 CCCCAGC 2223. CAGCCCAG 2224. DMSO 1 4 1 51 GCAGCACCGGG CCCCAC GCCACAGC CAGTTTC TTCA

Sanger Sequencing for Quantifying Frequencies of Indel Mutations

Purified PCR products used for T7EI assay were ligated into a Zero Blunt TOPO vector (Life Technologies) and transformed into chemically competent Top 10 bacterial cells. Plasmid DNAs were isolated and sequenced by the Massachusetts General Hospital (MGH) DNA Automation Core, using an M13 forward primer (5′-GTAAAACGACGGCCAG-3′) (SEQ ID NO:1059).

Restriction Digest Assay for Quantifying Specific Alterations Induced by HDR with ssODNs

PCR reactions of specific on-target sites were performed using Phusion high-fidelity DNA polymerase (New England Biolabs). The VEGF and EMX1 loci were amplified using a touchdown PCR program ((98° C., 10 s; 72-62° C., −1° C./cycle, 15 s; 72° C., 30 s)×10 cycles, (98° C., 10 s; 62° C., 15 s; 72° C., 30 s)×25 cycles), with 3% DMSO. The primers used for these PCR reactions are listed in Table E. PCR products were purified by Ampure XP beads (Agencourt) according to the manufacturer's instructions. For detection of the BamHI restriction site encoded by the ssODN donor template, 200 ng of purified PCR products were digested with BamHI at 37° C. for 45 minutes. The digested products were purified by Ampure XP beads (Agencourt), eluted in 20 ul 0.1×EB buffer and analyzed and quantified using a QIAXCEL capillary electrophoresis system.

TruSeq Library Generation and Sequencing Data Analysis

Locus-specific primers were designed to flank on-target and potential and verified off-target sites to produce PCR products ˜300 bp to 400 bps in length. Genomic DNAs from the pooled duplicate samples described above were used as templates for PCR. All PCR products were purified by Ampure XP beads (Agencourt) per the manufacturer's instructions. Purified PCR products were quantified on a QIAXCEL capillary electrophoresis system. PCR products for each locus were amplified from each of the pooled duplicate samples (described above), purified, quantified, and then pooled together in equal quantities for deep sequencing. Pooled amplicons were ligated with dual-indexed Illumina TruSeq adaptors as previously described (Fisher et al., 2011). The libraries were purified and run on a QIAXCEL capillary electrophoresis system to verify change in size following adaptor ligation. The adapter-ligated libraries were quantified by qPCR and then sequenced using Illumina MiSeq 250 bp paired-end reads performed by the Dana-Farber Cancer Institute Molecular Biology Core Facilities. We analyzed between 75,000 and 1,270,000 (average ˜422,000) reads for each sample. The TruSeq reads were analyzed for rates of indel mutagenesis as previously described (Sander et al., 2013). Specificity ratios were calculated as the ratio of observed mutagenesis at an on-target locus to that of a particular off-target locus as determined by deep sequencing. Fold-improvements in specificity with tru-RGNs for individual off-target sites were calculated as the specificity ratio observed with tru-gRNAs to the specificity ratio for that same target with the matched full-length gRNA. As mentioned in the text, for some of the off-target sites, no indel mutations were detected with tru-gRNAs. In these cases, we used a Poisson calculator to determine with a 95% confidence that the upper limit of the actual number of mutated sequences would be three in number. We then used this upper bound to estimate the minimum fold-improvement in specificity for these off-target sites.

Example 2a Truncated gRNAs can Efficiently Direct Cas9-Mediated Genome Editing in Human Cells

To test the hypothesis that gRNAs truncated at their 5′ end might function as efficiently as their full-length counterparts, a series of progressively shorter gRNAs were initially constructed as described above for a single target site in the EGFP reporter gene, with the following sequence: 5′-GGCGAGGGCGATGCCACCTAcGG-3′ (SEQ ID NO:2241). This particular EGFP site was chosen because it was possible to make gRNAs to it with 15, 17, 19, and 20 nts of complementarity that each have a G at their 5′ end (required for efficient expression from the U6 promoter used in these experiments). Using a human cell-based reporter assay in which the frequency of RGN-induced indels could be quantified by assessing disruption of a single integrated and constitutively expressed enhanced green fluorescent protein (EGFP) gene (Example 1 and Fu et al., 2013; Reyon et al., 2012) (FIG. 2B), the abilities of these variable-length gRNAs to direct Cas9-induced indels at the target site were measured.

As noted above, gRNAs bearing longer lengths of complementarity (21, 23, and 25 nts) exhibit decreased activities relative to the standard full-length gRNA containing 20 nts of complementary sequence (FIG. 2H), a result that matches those recently reported by others (Ran et al., Cell 2013). However, gRNAs bearing 17 or 19 nts of target complementarity showed activities comparable to or higher than the full-length gRNA, while a shorter gRNA bearing only 15 nts of complementary failed to show significant activity (FIG. 2H).

To test the generality of these initial findings, full-length gRNAs and matched gRNAs bearing 18, 17 and/or 16 nts of complementarity to four additional EGFP reporter gene sites (EGFP sites #1, #2, #3, and #4; FIG. 3A) were assayed. At all four target sites, gRNAs bearing 17 and/or 18 nts of complementarity functioned as efficiently as (or, in one case, more efficiently than) their matched full-length gRNAs to induce Cas9-mediated disruption of EGFP expression (FIG. 3A). However, gRNAs with only 16 nts of complementarity showed significantly decreased or undetectable activities on the two sites for which they could be made (FIG. 3A). For each of the different sites tested, we transfected the same amounts of the full-length or shortened gRNA expression plasmid and Cas9 expression plasmid. Control experiments in which we varied the amounts of Cas9 and truncated gRNA expression plasmids transfected for EGFP sites #1, #2, and #3 suggested that shortened gRNAs function equivalently to their full-length counterparts (FIGS. 3E (bottom) and 3F (bottom)) and that therefore we could use the same amounts of plasmids when making comparisons at any given target site. Taken together, these results provide evidence that shortened gRNAs bearing 17 or 18 nts of complementarity can generally function as efficiently as full-length gRNAs and hereafter the truncated gRNAs with these complementarity lengths are referred to as “tru-gRNAs” and RGNs using these tru-gRNAs as “tru-RGNs”.

Whether tru-RGNs could efficiently induce indels on chromatinized endogenous gene targets was tested next. Tru-gRNAs were constructed for seven sites in three endogenous human genes (VEGFA, EMXJ, and CLTA), including four sites that had previously been targeted with standard full-length gRNAs in three endogenous human genes: VEGFA site 1, VEGFA site 3, Erna, and CTLA (Example 1 and Fu et al., 2013; Hsu et al., 2013; Pattanayak et al., 2013) (FIG. 3B). (It was not possible to test a tru-gRNA for VEGFA site 2 from Example 1, because this target sequence does not have the G at either position 17 or 18 of the complementarity region required for gRNA expression from a U6 promoter.) Using a well-established T7 Endonuclease I (T7EI) genotyping assay (Reyon et al., 2012) as described above, the Cas9-mediated indel mutation frequencies induced by each of these various gRNAs at their respective target sites was quantified in human U2OS.EGFP cells. For all five of the seven four sites, tru-RGNs robustly induced indel mutations with efficiencies comparable to those mediated by matched standard RGNs (FIG. 3B). For the two sites on which tru-RGNs showed lower activities than their full-length counterparts, we note that the absolute rates of mutagenesis were still high (means of 13.3% and 16.6%) at levels that would be useful for most applications. Sanger sequencing for three of these target sites (VEGFA sites 1 and 3 and EMU) confirmed that indels induced by tru-RGNs originate at the expected site of cleavage and that these mutations are essentially indistinguishable from those induced with standard RGNs (FIG. 3C and FIGS. 7A-D).

We also found that tru-gRNAs bearing a mismatched 5′ G and an 18 nt complementarity region could efficiently direct Cas9-induced indels whereas those bearing a mismatched 5′ G and a 17 nt complementarity region showed lower or undetectable activities compared with matched full-length gRNAs (FIG. 7E), consistent with our findings that a minimum of 17 nts of complementarity is required for efficient RGN activity.

To further assess the genome-editing capabilities of tru-RGNs, their abilities to induce precise sequence alterations via HDR with ssODN donor templates were tested. Previous studies have shown that Cas9-induced breaks can stimulate the introduction of sequence from a homologous ssODN donor into an endogenous locus in human cells (Cong et al., 2013; Mali et al., 2013c; Ran et al., 2013; Yang et al., 2013). Therefore, the abilities were compared of matched full-length and tru-gRNAs targeted to VEGFA site 1 and to the Erna site to introduce a BamHI restriction site encoded on homologous ssODNs into these endogenous genes. At both sites, tru-RGNs mediated introduction of the BamHI site with efficiencies comparable to those seen with standard RGNs harboring their full-length gRNA counterparts (FIG. 3D). Taken together, this data demonstrate that tru-RGNs can function as efficiently as standard RGNs to direct both indels and precise HDR-mediated genome editing events in human cells.

Example 2b Tru-RGNs Exhibit Enhanced Sensitivities to gRNA/DNA Interface Mismatches

Having established that tru-RGNs can function efficiently to induce on-target genome editing alterations, whether these nucleases would show greater sensitivity to mismatches at the gRNA/DNA interface was tested. To assess this, a systematic series of variants was constructed for the tru-gRNAs that were previously tested on EGFP sites #1, #2, and #3 (FIG. 3A above). The variant gRNAs harbor single Watson-Crick substitutions at each position within the complementarity region (with the exception of the 5′ G required for expression from the U6 promoter) (FIG. 5A). The human cell-based EGFP disruption assay was used to assess the relative abilities of these variant tru-gRNAs and an analogous set of matched variant full-length gRNAs made to the same three sites as described in Example 1 to direct Cas9-mediated indels. The results show that for all three EGFP target sites, tru-RGNs generally showed greater sensitivities to single mismatches than standard RGNs harboring matched full-length gRNAs (compare bottom and top panels of FIG. 5A). The magnitude of sensitivity varied by site, with the greatest differences observed for sites #2 and #3, whose tru-gRNAs harbored 17 nts of complementarity.

Encouraged by the increased sensitivity of tru-RGNs to single nucleotide mismatches, we next sought to examine the effects of systematically mismatching two adjacent positions at the gRNA-DNA interface. We therefore made variants of the tru-gRNAs targeted to EGFP target sites #1, #2, and #3, each bearing Watson-Crick transversion substitutions at two adjacent nucleotide positions (FIG. 5B). As judged by the EGFP disruption assay, the effects of adjacent double mismatches on RGN activity were again substantially greater for tru-gRNAs than for the analogous variants made in Example 1 for matched full-length gRNAs targeted to all three EGFP target sites (compare bottom to top panels in FIG. 5B). These effects appeared to be site-dependent with nearly all of the double-mismatched tru-gRNAs for EGFP sites #2 and #3 failing to show an increase in EGFP disruption activities relative to a control gRNA lacking a complementarity region and with only three of the mismatched tru-gRNA variants for EGFP site #1 showing any residual activities (FIG. 5B). In addition, although double mutations generally showed greater effects on the 5′ end with full-length gRNAs, this effect was not observed with tru-gRNAs. Taken together, our data suggest that tru-gRNAs exhibit greater sensitivities than full-length gRNAs to single and double Watson-Crick transversion mismatches at the gRNA-DNA interface.

Example 2c Tru-RGNs Targeted to Endogenous Genes Show Improved Specificities in Human Cells

The next experiments were performed to determine whether tru-RGNs might show reduced genomic off-target effects in human cells relative to standard RGNs harboring full-length gRNA counterparts. We examined matched full-length and tru-gRNAs targeted to VEGFA site 1, VEGFA site 3, and EMX1 site 1 (described in FIG. 3B above) because previous studies (see Example 1 and Fu et al., 2013; Hsu et al., 2013) had defined 13 bona fide off-target sites for the full-length gRNAs targeted to these sites. (We were unable to test a tru-gRNA for VEGFA site 2 from our original study6 because this target sequence does not have the G at either position 17 or 18 of the complementarity region required for efficient gRNA expression from a U6 promoter.) Strikingly, we found that tru-RGNs showed substantially reduced mutagenesis activity in human U2OS.EGFP cells relative to matched standard RGNs at all 13 of these bona fide off-target sites as judged by T7EI assay (Table 3A); for 11 of the 13 off-target sites, the mutation frequency with tru-RGNs dropped below the reliable detection limit of the T7EI assay (2-5%) (Table 3A). We observed similar results when these matched pairs of standard and tru-RGNs were tested at the same 13 off-target sites in another human cell line (FT-HEK293 cells) (Table 3A).

To quantify the magnitude of specificity improvement observed with tru-RGNs, we measured off-target mutation frequencies using high-throughput sequencing, which provides a more sensitive method for detecting and quantifying low frequency mutations than the T7EI assay. We assessed a subset of 12 of the 13 bona fide off-target sites for which we had seen decreased mutation rates with tru-gRNAs by T7EI assay (for technical reasons, we were unable to amplify the required shorter amplicon for one of the sites) and also examined an additional off-target site for EMX1 site 1 that had been identified by another group? (FIG. 6A). For all 13 off-target sites we tested, tru-RGNs showed substantially decreased absolute frequencies of mutagenesis relative to matched standard RGNs (FIG. 6A and Table 3B) and yielded improvements in specificity of as much as ˜5000-fold or more relative to their standard RGN counterparts (FIG. 6B). For two off-target sites (OT1-4 and OT1-11), it was difficult to quantify the on-target to off-target ratios for tru-RGNs because the absolute number and frequency of indel mutations induced by tru-RGNs fell to background or near-background levels. Thus, the ratio of on-target to off-target rates would calculate to be infinite in these cases. To address this, we instead identified the maximum likely indel frequency with a 95% confidence level for these sites and then used this conservative estimate to calculate the minimum likely magnitude of specificity improvement for tru-RGNs relative to standard RGNs for these off-targets. These calculations suggest tru-RGNs yield improvements of ˜10,000-fold or more at these sites (FIG. 6B).

To further explore the specificity of tru-RGNs, we examined their abilities to induce off-target mutations at additional closely related sites in the human genome. For the tru-gRNAs to VEGFA site 1 and Erna, which each possess 18 nts of target site complementarity, we computationally identified all additional sites in the human genome mismatched at one or two positions within the complementarity region (not already examined above in Table 3A) and a subset of all sites mismatched at three positions among which we favored mismatches in the 5′ end of the site as described in Example 1. For the tru-gRNA to VEGFA site 3, which possesses 17 nts of target site complementarity, we identified all sites mismatched at one position and a subset of all sites mismatched at two positions among which mismatches in the 5′ end were favored (again not already examined in Table 3A). This computational analysis yielded a total of 30, 30, and 34 additional potential off-target sites for the tru-RGNs targeted to VEGFA site 1, VEFGA site 3, and the EMX1 site, respectively, which we then assessed for mutations using T7EI assay in human U2OS.EGFP and HEK293 cells in which the RGNs had been expressed.

Strikingly, the three tru-RGNs to VEGFA site 1, VEFGA site 3, and EMX1 did not induce detectable Cas9-mediated indel mutations at 93 of the 94 potential off-target sites examined in human U2OS.EGFP cells or at any of the 94 potential off-target sites in human HEK293 cells (Table 3C). For the one site at which off-target mutations were seen, whether the standard RGN with a full-length gRNA targeted to VEGFA site 1 could also mutagenize this same off-target site was examined; it induced detectable mutations albeit at a slightly lower frequency (FIG. 6C). The lack of improvement observed with shortening of the gRNA at this off-target site can be understood by comparing the 20 and 18 nt sequences for the full-length and tru-gRNAs, which shows that the two additional bases in the full-length 20 nt target are both mismatched (FIG. 6C). In summary, based on this survey of 94 additional potential off-target sites, shortening of the gRNA does not appear to induce new high-frequency off-target mutations.

Deep sequencing of a subset of the 30 most closely matched potential off-target sites from this set of 94 site (i.e.—those with one or two mismatches) showed either undetectable or very low rates of indel mutations (Table 3D) comparable to what we observed at other previously identified off-target sites (Table 3B). We conclude that tru-RGNs generally appear to induce either very low or undetectable levels of mutations at sites that differ by one or two mismatches from the on-target site. This contrasts with standard RGNs for which it was relatively easy to find high-frequency off-target mutations at sites that differed by as many as five mismatches (see Example 1).

TABLE 3A On- and off-target mutation frequencies of matched tru-RGNs and standard RGNs targeted to endogenous genes in human U2OS.EGFP and HEK293 cells Indel mutation Indel mutation Tar- SEQ frequency SEQ frequency get 20mer ID (%) ± s.e.m. Truncated ID (%) ± s.e.m. ID Target NO: U2OS.EGFP HEK293 Target NO: U2OS.EGFP HEK293 Gene T1 GGGTGGGGGGAG 2242. 23.69 ±  6.98 ± GTGGGGGGAGT 2243. 23.93 ±  8.34 ± VEGFA TTTGCTCCtGG  1.99  1.33 TTGCTCCtGG  4.37  0.01 OT1-3 GG A TGG A GGGAG 2244. 17.25 ±  7.26 ± A TGG A GGGAGT 2245. N.D. N.D. IGDCC3 TTTGCTCCtGG  2.97  0.62 TTGCTCCtGG OT1-4 GGG A GGG T GGAG 2246.  6.23 ±  2.66 ± G A GGG T GGAGT 2247. N.D. N.D. LOC116437 TTTGCTCCtGG  0.20  0.30 TTGCTCCtGG OT1-6 C GG G GG A GGGAG 2248.  3.73 ±  1.41 ± G G GG A GGGAGT 2249. N.D. N.D. CACNA2D TTTGCTCCtGG  0.23  0.07 TTGCTCCtGG OT1-11 GGG GA GGGG A AG 2250. 10.4 ±  3.61 ± G GA GGGG A AGT 2251. N.D. N.D. TTTGCTCCtGG  0.7  0.02 TTGCTCCtGG T3 GGTGAGTGAGTG 2252. 54.08 ± 22.97 ± GAGTGAGTGTG 2253. 50.49 ± 20.05 ± VEGFA TGTGCGTGtGG  1.02  0.17 TGCGTGtGG  1.25  0.01 OT3-1 GGTGAGTGAGTG 2254.  6.16 ±  6.02 ± GAGTGAGTGTG 2255. N.D. N.D. (abParts) TGTG T GTGaGG  0.98  0.11 TG T GTGaGG OT3-2 A GTGAGTGAGTG 2256. 19.64 ± 11.29 ± GAGTGAGTGTG 2257.  5.52 ±  3.41 ± MAX TGTG T GTGgGG  1.06  0.27 TG T GTGgGG  0.25  0.07 OT3-4 G C TGAGTGAGTG 2258.  7.95 ±  4.50 ± GAGTGAGTGT A 2259.  1.69 ±  1.27 ± T A TGCGTGtGG  0.11  0.02 TGCGTGtGG  0.26  0.10 OT3-9 GGTGAGTGAGTG 2260. N.D.  1.09 ± GAGTGAGTG C G 2261. N.D. N.D. TPCN2 C GTGCG G GtGG  0.17 TGCG G GtGG OT3-17 G T TGAGTGA A TG 2262.  1.85 ± N.D. GAGTGA A TGTG 2263. N.D. N.D. SLIT1 TGTGCGTGaGG  0.08 TGCGTGaGG OT3-18 T GTG G GTGAGTG 2264.  6.16 ±  6.27 ± G G GTGAGTGTG 2265. N.D. N.D. COMDA TGTGCGTGaGG  0.56  0.09 TGCGTGaGG OT3-20 A G A GAGTGAGTG 2266. 10.47 ±  4.38 ± GAGTGAGTGTG 2267. N.D. N.D. TGTGC A TGaGG  1.08  0.58 TGC A TGaGG T4 GAGTCCGAGCAG 2268. 41.56 ± 12.65 ± GTCCGAGCAGA 2269. 43.01 ± 17.25 ± EMX1 AAGAAGAAgGG  0.20  0.31 AGAAGAAgGG  0.87  0.64 OT4-1 GAGT TA GAGCAG 2270. 19.26 ±  4.14 ± GT TA GAGCAGA 2271. N.D. N.D. HCN1 AAGAAGAAaGG  0.73  0.66 AGAAGAAaGG OT- GAGTC TA AGCAG 2272.  4.37 ± N.D. GTC TA AGCAGA 2273. N.D. N.D. MFAP1 4_Hsu31 AAGAAGAAg A G  0.58 AGAAGAAg A G Mutation frequencies were measured by T7EI assay. Means of duplicate measurements are shown with error bars representing standard errors of the mean. *Off-target site OT4_53 is the same as EMX1 target 3 OT31 from Hsu et al., 2013.

TABLE 3B Numbers of wild-type (WT) and indel mutation sequencing reads from deep sequencing experiments Control tru-RGN Standard RGN Site Indel WT Freq. Indel WT Freq. Indel WT Freq. VEGFA site 1 45 140169 0.03% 122858 242127 33.66% 150652 410479 26.85% OT1-3 0 132152 0.00% 1595 205878 0.77% 50973 144895 26.02% OT1-4 0 133508 0.00% 0 223881 0.00% 22385 240873 8.50% OT1-6 3 213642 0.00% 339 393124 0.09% 24332 424458 5.21% OT1-11 1 930894 0.00% 0 274779 0.00% 43738 212212 17.09% VEGFA site 3 5 212571 0.00% 303913 292413 50.96% 183626 174740 51.24% OT3-2 1169 162545 0.71% 9415 277616 3.28% 26545 222482 10.66% OT3-4 7 383006 0.00% 15551 1135673 1.35% 42699 546203 7.25% OT3-9 73 145367 0.05% 113 227874 0.05% 1923 168293 1.13% OT3-17 8 460498 0.00% 31 1271276 0.00% 16760 675708 2.42% OT3-18 7 373571 0.00% 284 1275982 0.02% 72354 599030 10.78% OT3-20 5 140848 0.00% 593 325162 0.18% 30486 202733 13.07% EMX1 site 1 1 158838 0.00% 49104 102805 32.32% 128307 307584 29.44% OT4-1 10 169476 0.01% 13 234039 0.01% 47426 125683 27.40% OT4-52 2 75156 0.00% 10 231090 0.00% 429 340201 0.13% OT4-53 0 234069 0.00% 6 367811 0.00% 17421 351667 4.72% Freq. = frequency of indel mutations = number of indel sequences/number of wild-type sequences. Control gRNA = gRNA lacking a complementarity region

TABLE 3C Indel mutation frequencies at potential off-target sites of  tru-RGNs targeted to endogenous genes in human cells Indel mutation frequency SEQ (%) ± s.e.m. Target ID Number of U2OS.EGFP ID Target Site + PAM NO: mismatches cells HEK293 cells VEGFA GTGGGGGGAGTTTGCTCCtGG 2274. 0 23.93 ± 4.37   8.34 ± 0.01 Site 1 (on-target) GTGGGGGGAGTTTGCCCCaGG 2275. 1 Not detected Not detected GTGGGGGGTGTTTGCTCCcGG 2276. 1 Not detected Not detected GTGGGTGGAGTTTGCTACtGG 2277. 2 Not detected Not detected GTGGGGGGAGCTTTCTCCtGG 2278. 2 Not detected Not detected GTGGGTGGCGTTTGCTCCaGG 2279. 2 Not detected Not detected GTGGAGGGAGCTTGCTCCtGG 2280. 2  6.88 ± 0.19 Not detected GTGGGTGGAGTTTGCTACaGG 2281. 2 Not detected Not detected GGGGGGGCAGTTTGCTCCtGG 2282. 2 Not detected Not detected GTGTGGGGAATTTGCTCCaGG 2283. 2 Not detected Not detected CTGCTGGGAGTTTGCTCCtGG 2284. 3 Not detected Not detected TTTGGGAGAGTTTGCTCCaGG 2285. 3 Not detected Not detected CTGAGGGCAGTTTGCTCCaGG 2286. 3 Not detected Not detected GTAAGGGAAGTTTGCTCCtGG 2287. 3 Not detected Not detected GGGGGTAGAGTTTGCTCCaGG 2288. 3 Not detected Not detected GGGTGGGGACTTTGCTCCaGG 2289. 3 Not detected Not detected GGGGGAGCAGTTTGCTCCaGG 2290. 3 Not detected Not detected TTGGGGTTAGTTTGCTCCtGG 2291. 3 Not detected Not detected TTGAGGGGAGTCTGCTCCaGG 2292. 3 Not detected Not detected CTGGGGTGATTTTGCTCCtGG 2293. 3 Not detected Not detected GAGAGGGGAGTTGGCTCCtGG 2294. 3 Not detected Not detected TTTGGGGGAGTTTGCCCCaGG 2295. 3 Not detected Not detected TTCGGGGGAGTTTGCGCCgGG 2296. 3 Not detected Not detected CTCGGGGGAGTTTGCACCaGG 2297. 3 Not detected Not detected GTGTTGGGAGTCTGCTCCaGG 2298. 3 Not detected Not detected GAGGGGGCAGGTTGCTCCaGG 2299. 3 Not detected Not detected GAGGGGAGAGTTTGTTCCaGG 2300. 3 Not detected Not detected GTGGCTGGAGTTTGCTGCtGG 2301. 3 Not detected Not detected GTCGGGGGAGTGGGCTCCaGG 2302. 3 Not detected Not detected GAGGGGGGAGTGTGTTCCgGG 2303. 3 Not detected Not detected GTGGTGGGAGCTTGTTCCtGG 2304. 3 Not detected Not detected GTGGGGGGTGCCTGCTCCaGG 2305. 3 Not detected Not detected VEGFA GAGTGAGTGTGTGCGTGtGG 2306. 0 50.49 ± 1.25 20.05 ± 0.01 Site 3 (on-target) CAGTGAGTGTGTGCGTGtGG 2307. 1 Not detected Not detected GTGTGAGTGTGTGCGTGgGG 2308. 1 Not detected Not detected GTGTGAGTGTGTGCGTGaGG 2309. 1 Not detected Not detected GTGTGAGTGTGTGCGTGtGG 2310. 1 Not detected Not detected GAGTGTGTGTGTGCGTGtGG 2311. 1 Not detected Not detected GAGTGGGTGTGTGCGTGgGG 2312. 1 Not detected Not detected GAGTGACTGTGTGCGTGtGG 2313. 1 Not detected Not detected GAGTGAGTGTGTGGGTGgGG 2314. 1 Not detected Not detected GAGTGAGTGTGTGTGTGtGG 2315. 1 Not detected Not detected GAGTGAGTGTGTGTGTGtGG 2316. 1 Not detected Not detected GAGTGAGTGTGTGTGTGgGG 2317. 1 Not detected Not detected GAGTGAGTGTGTGTGTGtGG 2318. 1 Not detected Not detected GAGTGAGTGTGTGCGCGgGG 2319. 1 Not detected Not detected CTGTGAGTGTGTGCGTGaGG 2320. 2 Not detected Not detected ATGTGAGTGTGTGCGTGtGG 2321. 2 Not detected Not detected GCCTGAGTGTGTGCGTGtGG 2322. 2 Not detected Not detected GTGTGTGTGTGTGCGTGtGG 2323. 2 Not detected Not detected GTGTGGGTGTGTGCGTGtGG 2324. 2 Not detected Not detected GCGTGTGTGTGTGCGTGtGG 2325. 2 Not detected Not detected GTGTGTGTGTGTGCGTGgGG 2326. 2 Not detected Not detected GTGTGCGTGTGTGCGTGtGG 2327. 2 Not detected Not detected GTGTGTGTGTGTGCGTGcGG 2328. 2 Not detected Not detected GAGAGAGAGTGTGCGTGtGG 2329. 2 Not detected Not detected GAGTGTGTGAGTGCGTGgGG 2330. 2 Not detected Not detected GTGTGAGTGTGTGTGTGtGG 2331. 2 Not detected Not detected GAGTGTGTGTATGCGTGtGG 2332. 2 Not detected Not detected GAGTCAGTGTGTGAGTGaGG 2333. 2 Not detected Not detected GAGTGTGTGTGTGAGTGtGG 2334. 2 Not detected Not detected GAGTGTGTGTGTGCATGtGG 2335. 2 Not detected Not detected GAGTGAGAGTGTGTGTGtGG 2336. 2 Not detected Not detected GAGTGAGTGAGTGAGTGaGG 2337. 2 Not detected Not detected EMX1  GTCCGAGCAGAAGAAGAAgGG 2338. 0 43.01 ± 0.87 17.25 ± 0.64 site (on-target) GTCTGAGCAGAAGAAGAAtGG 2339. 1 Not detected Not detected GTCCCAGCAGTAGAAGAAtGG 2340. 2 Not detected Not detected GTCCGAGGAGAGGAAGAAaGG 2341. 2 Not detected Not detected GTCAGAGGAGAAGAAGAAgGG 2342. 2 Not detected Not detected GACAGAGCAGAAGAAGAAgGG 2343. 2 Not detected Not detected GTGGGAGCAGAAGAAGAAgGG 2344. 2 Not detected Not detected GTACTAGCAGAAGAAGAAaGG 2345. 2 Not detected Not detected GTCTGAGCACAAGAAGAAtGG 2346. 2 Not detected Not detected GTGCTAGCAGAAGAAGAAgGG 2347. 2 Not detected Not detected TACAGAGCAGAAGAAGAAtGG 2348. 3 Not detected Not detected TACGGAGCAGAAGAAGAAtGG 2349. 3 Not detected Not detected AACGGAGCAGAAGAAGAAaGG 2350. 3 Not detected Not detected GACACAGCAGAAGAAGAAgGG 2351. 3 Not detected Not detected CTGCGATCAGAAGAAGAAaGG 2352. 3 Not detected Not detected GACTGGGCAGAAGAAGAAgGG 2353. 3 Not detected Not detected TTCCCTGCAGAAGAAGAAaGG 2354. 3 Not detected Not detected TTCCTACCAGAAGAAGAAtGG 2355. 3 Not detected Not detected CTCTGAGGAGAAGAAGAAaGG 2356. 3 Not detected Not detected ATCCAATCAGAAGAAGAAgGG 2357. 3 Not detected Not detected GCCCCTGCAGAAGAAGAAcGG 2358. 3 Not detected Not detected ATCCAACCAGAAGAAGAAaGG 2359. 3 Not detected Not detected GACTGAGAAGAAGAAGAAaGG 2360. 3 Not detected Not detected GTGGGATCAGAAGAAGAAaGG 2361. 3 Not detected Not detected GACAGAGAAGAAGAAGAAaGG 2362. 3 Not detected Not detected GTCATGGCAGAAGAAGAAaGG 2363. 3 Not detected Not detected GTTGGAGAAGAAGAAGAAgGG 2364. 3 Not detected Not detected GTAAGAGAAGAAGAAGAAgGG 2365. 3 Not detected Not detected CTCCTAGCAAAAGAAGAAtGG 2366. 3 Not detected Not detected TTCAGAGCAGGAGAAGAAtGG 2367. 3 Not detected Not detected GTTGGAGCAGGAGAAGAAgGG 2368. 3 Not detected Not detected GCCTGAGCAGAAGGAGAAgGG 2369. 3 Not detected Not detected GTCTGAGGACAAGAAGAAtGG 2370. 3 Not detected Not detected GTCCGGGAAGGAGAAGAAaGG 2371. 3 Not detected Not detected GGCCGAGCAGAAGAAAGAcGG 2372. 3 Not detected Not detected GTCCTAGCAGGAGAAGAAgAG 2373. 3 Not detected Not detected

TABLE 3D Frequencies of tru-RGN-induced indel mutations at potential off- target sites in human U2OS.EGFP as determined by deep sequencing On- target Off-target site tru-RGN Control site sequence S# Indel WT Freq. Indel WT Freq VEGFA GTGGGGGGAGTTTGC C CCaGG 2374. 150 225640 0.66%   3 135451 0.00% site 1   0 GTGGGGGG T GTTTGCTCCcGG 2375. 155 152386 1.01%   0  86206 0.00%   2 GTGGG T GGAGTTTGCT A CtGG 2376.   1 471818 0.00%   0 199581 0.00% GTGGG T GGAGTTTGCT A CaGG 2377.   0 337298 0.00%   1 211547 0.00% GTGGG T GG C GTTTGCTCCaGG 2378.   2 210174 0.00%   1 105531 0.00% GTG T GGGGA A TTTGCTCCaGG 2379. 673 715547 0.09%   1 387097 0.00% GTGGGGGGAG C TT T CTCCtGG 2380.   5 107757 0.00%   1  58735 0.00% G G GGGGG C AGTTTGCTCCtGG 2381. 191 566548 0.34%   3 297083 0.00%   4 VEGFA G T GTGAGTGTGTGCGTGtGG 2382.  58 324881 0.02%   9 122216 0.01% site 3 G T GTGAGTGTGTGCGTGaGG 2383. 532 194914 0.27%  11  73644 0.01% GAGTG G GTGTGTGCGTGgGG 2384.  70 237029 0.03%  10 178258 0.01% GAGTGA C TGTGTGCGTGtGG 2385.   6 391894 0.00%   0 239460 0.00% GAGTGAGTGTGTG G GTGgGG 2386.  15 160140 0.01%  10 123324 0.01% G T GTGAGTGTGTGCGTGgGG 2387.  19 138687 0.01%   1 196271 0.00% C AGTGAGTGTGTGCGTGtGG 2388.  78 546865 0.01%  41 355953 0.01% G T GTGAGTGTGTGCGTGtGG 2389. 128 377451 0.03%  56 133978 0.04% GAGTG T GTGTGTGCGTGtGG 2390. 913 263028 0.35%  78 178979 0.04% GAGTGAGTGTGTG T GTGtGG 2391.  40 106933 0.04%  36  58812 0.06% GAGTGAGTGTGTG T GTGtGG 2392. 681 762999 0.09%  63 222451 0.03% GAGTGAGTGTGTG T GTGgGG 2393. 331 220289 0.15% 100 113911 0.09% GAGTGAGTGTGTG T GTGtGG 2394.   0 35725 0.00%   8 186495 0.00% GAGTGAGTGTGTGCG C GgGG 2395.  94 246893 0.04%  16 107623 0.01% EMX1 GTC A GAG G AGAAGAAGAAgGG 2396.   0 201483 0.00%   4 148416 0.00% site 1 GTC A GAG G AGAAGAAGAAgGG 2397.  10 545662 0.00%   5 390884 0.00% GTC T GAGCA C AAGAAGAAtGG 2398.   2 274212 0.00%   0 193837 0.00% GTC T GAGCAGAAGAAGAAtGG 2399. 440 375646 0.12%  10 256181 0.00% G A C A GAGCAGAAGAAGAAgGG 2400.   2 212472 0.00%   1 158860 0.00% GT A C T AGCAGAAGAAGAAaGG 2401. 152 229209 0.07% 103 157717 0.07% GT GG GAGCAGAAGAAGAAgGG 2402.  50 207401 0.02%  36 111183 0.03% GTCC C AGCAG T AGAAGAAtGG 2403.   0 226477 0.00%   1 278948 0.00% S#: SEQ ID NO:

Example 2d Tru-gRNAs can be Used with Dual Cas9 Nickases to Efficiently Induce Genome Editing in Human Cells

tru-gRNAs were tested with the recently described dual Cas9 nickase approach to induce indel mutations. To do this, the Cas9-D10A nickase together with two full-length gRNAs targeted to sites in the human VEGFA gene (VEGFA site 1 and an additional sequence we refer to as VEGFA site 4) were co-expressed in U2OS.EGFP cells (FIG. 4A). As described previously (Ran et al., 2013), this pair of nickases functioned cooperatively to induce high rates of indel mutations at the VEGFA target locus (FIG. 4B). Interestingly, Cas9-D10A nickase co-expressed with only the gRNA targeted to VEGFA site 4 also induced indel mutations at a high frequency, albeit at a rate somewhat lower than that observed with the paired full-length gRNAs (FIG. 4B). Importantly, use of a tru-gRNA for VEGFA site 1 in place of a full-length gRNA did not affect the efficacy of the dual nickase approach to induce indel mutations (FIG. 4B).

The dual nickase strategy has also been used to stimulate the introduction of specific sequence changes using ssODNs (Mali et al., 2013a; Ran et al., 2013) and so whether tru-gRNAs might be used for this type of alteration was also tested. Paired full-length gRNAs for VEGFA sites 1 and 4 together with Cas9-D10A nickase cooperatively enhanced efficient introduction of a short insertion from a ssODN donor (FIG. 3A) into the VEGFA locus in human U2OS.EGFP cells as expected (FIG. 3C). Again, the efficiency of ssODN-mediated sequence alteration by dual nicking remained equally high with the use of a tru-gRNA in place of the full-length gRNA targeted to VEGFA site 1 (FIG. 3C). Taken together, these results demonstrate that tru-gRNAs can be utilized as part of a dual Cas9 nickase strategy to induce both indel mutations and ssODN-mediated sequence changes, without compromising the efficiency of genome editing by this approach.

Having established that use of a tru-gRNA does not diminish the on-target genome editing activities of paired nickases, we next used deep sequencing to examine mutation frequencies at four previously identified bona fide off-target sites of the VEGFA site 1 gRNA. This analysis revealed that mutation rates dropped to essentially undetectable levels at all four of these off-target sites when using paired nickases with a tru-gRNA (Table 4). By contrast, neither a tru-RGN (Table 3B) nor the paired nickases with full-length gRNAs (Table 4) was able to completely eliminate off-target mutations at one of these four off-target sites (OT1-3). These results demonstrate that the use of tru-gRNAs can further reduce the off-target effects of paired Cas9 nickases (and vice versa) without compromising the efficiency of on-target genome editing.

TABLE 4 Frequencies of paired nickase-induced indel mutations at on- and off-target sites of VEGFA site 1 using full-length and tru-gRNAs Paired full-length gRNAs tru-gRNA/full-length gRNA Control Site Indel WT Freq. Indel WT Freq. Indel WT Freq. VEGFA site 1 78905 345696 18.583% 65754 280720 18.978% 170 308478 0.055% OT1-3 184 85151 0.216% 0 78658 0.000% 2 107850 0.002% OT1-4 0 89209 0.000% 1 97010 0.001% 0 102135 0.000% OT1-6 2 226575 0.001% 0 208218 0.000% 0 254580 0.000% OT1-11 0 124729 0.000% 0 121581 0.000% 0 155173 0.000%

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OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. 

What is claimed is:
 1. A method of increasing specificity of Streptococcus pyogenes CRISPR/Cas9 (Cas9) RNA-guided genome editing in a cell, the method comprising contacting the cell with a guide RNA that includes a complementarity region at the 5′ end of the guide RNA consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of a selected target genomic sequence, wherein the target sequence is immediately 5′ of a protospacer adjacent motif (PAM), and wherein the guide RNA is (i) a single guide RNA that includes at the 5′ end of the single guide RNA a complementarity region consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of the selected target genomic sequence on a double-stranded DNA molecule, or (ii) a crRNA that includes at the 5′ end of the crRNA a complementarity region consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of the selected target genomic sequence, and a tracrRNA, wherein in the presence of a S. pyogenes Cas9 genome editing enzyme, the guide RNA complementarity region binds and directs the Cas9 genome editing enzyme to the target genomic sequence, thereby increasing specificity of RNA-guided genome editing in a cell.
 2. The method of claim 1, wherein the crRNA is SEQ ID NO: 2407 and the tracrRNA is SEQ ID NO: 8; the crRNA is SEQ ID NO: 2404 and the tracrRNA is SEQ ID NO: 2405; or the crRNA is SEQ ID NO: 2408 and the tracrRNA is SEQ ID NO:
 2406. 3. The method of claim 1, wherein the tracrRNA is selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 2405, SEQ ID NO: 2406, SEQ ID NO: 2409, SEQ ID NO: 2410, SEQ ID NO: 2411 and SEQ ID NO:
 2412. 4. The method of claim 1, wherein the guide RNA is (i) the single guide RNA that includes at the 5′ end of the single RNA the complementarity region consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of the selected target genomic sequence.
 5. The method of claim 1, wherein the guide RNA is a ribonucleic acid selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 2404, SEQ ID NO: 2407 and SEQ ID NO:
 2408. 6. The method of claim 1, wherein the complementarity region is complementary to 17 consecutive nucleotides of the complementary strand of the selected target genomic sequence.
 7. The method of claim 1, wherein the complementarity region is complementary to 18 consecutive nucleotides of the complementary strand of the selected target genomic sequence.
 8. The method of claim 1, wherein the cell is a eukaryotic cell.
 9. A method of inducing a break in a target region of a double-stranded DNA molecule in a cell, the method comprising expressing in or introducing into the cell: a S. pyogenes CRISPR/Cas9 nuclease or nickase; and a guide RNA that includes a complementarity region at the 5′ end of the guide RNA consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of a double-stranded DNA molecule, wherein the target region sequence is immediately 5′ of a protospacer adjacent motif (PAM), and wherein the guide RNA complementarity region binds and directs the Cas9 nuclease or nickase to the target region sequence of a double-stranded DNA molecule, and wherein the guide RNA is (i) a single guide RNA that includes at the 5′ end of the single guide RNA a complementarity region consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of a selected target genomic sequence on a double stranded DNA molecule, or (ii) a crRNA that includes at the 5′ end of the crRNA a complementarity region consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of a selected target genomic sequence, and a tracrRNA; thereby inducing a break in the target region of a double-stranded DNA molecule in a cell.
 10. The method of claim 9, wherein the guide RNA comprises a ribonucleic acid selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 2404, SEQ ID NO: 2407 and SEQ ID NO:
 2408. 11. The method of claim 9, wherein the complementarity region is complementary to 17 consecutive nucleotides of the complementary strand of the selected target region of a double-stranded DNA molecule.
 12. The method of claim 9, wherein the complementarity region is complementary to 18 consecutive nucleotides of the complementary strand of a selected target region of the double-stranded DNA molecule.
 13. The method of claim 9, wherein the target region is in a target genomic sequence.
 14. The method of claim 9, wherein the cell is a eukaryotic cell.
 15. The method of claim 9, wherein the crRNA is SEQ ID NO: 2407 and the tracrRNA is SEQ ID NO: 8; the crRNA is SEQ ID NO: 2404 and the tracrRNA is SEQ ID NO: 2405; or the mRNA is SEQ ID NO: 2408 and the tracrRNA is SEQ ID NO:
 2406. 16. The method of claim 9, wherein the tracrRNA is selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 2405, SEQ ID NO: 2406, SEQ ID NO: 2409, SEQ ID NO: 2410, SEQ ID NO: 2411 and SEQ ID NO:
 2412. 17. A method of modifying a target region of a double-stranded DNA molecule in a cell, the method comprising expressing in or introducing into the cell: a S. pyogenes CRISPR dCas9-heterologous functional domain fusion protein (dCas9-HFD); and a guide RNA that includes a complementarity region at the 5′ end of the guide RNA consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of a selected target sequence present on a double-stranded DNA molecule, wherein the target sequence is immediately 5′ of a protospacer adjacent motif (PAM), and wherein the guide RNA is: (i) a single guide RNA that includes a complementarity region at the 5′ end of the single guide RNA consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of a selected target genomic sequence on a double stranded DNA molecule, or (ii) a crRNA that includes at the 5′ end of the crRNA a complementarity region consisting of 17-18 nucleotides that are complementary to 17-18 consecutive nucleotides of the complementary strand of a selected target genomic sequence, and a tracrRNA, and wherein the guide RNA complementarity region binds and directs the dCas9-HFD to the target region of the double-stranded DNA molecule, thereby modifying the target region of a double-stranded DNA molecule in a cell. 